Compositions, systems, and methods for generating inner ear hair cells for treatment of hearing loss

ABSTRACT

Method and compositions for inducing the self-renewal of stem/progenitor supporting cells comprised by a cochlear cell population, including inducing the stem/progenitor cells to proliferate while maintaining, in the daughter cells, the capacity to differentiate into hair cells.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/845,263, filed on Sep. 3, 2015, which claims the benefit of U.S.Provisional Application No. 62/045,506, filed on Sep. 3, 2014, and U.S.Provisional Application No. 62/051,003, filed on Sep. 16, 2014. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R01DE013023 awarded by the National Institutes of Health. This inventionwas made with government support under Grant No. HL095722 awarded by theNational Institute of Health. The Government has certain rights in theinvention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file:

-   -   a) File name: 00502282017_SEQUENCELISTING.txt; created Jan. 23,        2020, 2 KB in size.

BACKGROUND OF THE INVENTION

Permanent damage to the hair cells of the inner ear results insensorineural hearing loss, leading to communication difficulties in alarge percentage of the population. Hair cells are the receptor cellsthat transduce the acoustic stimulus. Regeneration of damaged hair cellsprovide an avenue for the treatment of a condition that currently has notherapies other than prosthetic devices. Although hair cells do notregenerate in the mammalian cochlea, new hair cells in lower vertebratesare generated from epithelial cells, called supporting cells, thatsurround hair cells.

The prevalence of hearing loss after damage to the mammalian cochlea hasbeen thought to be due to a lack of spontaneous regeneration of haircells and/or neurons, the primary components to detect sound (Wong andRyan, 2015). Humans are born with about 15,000 inner ear hair cells andhair cells do not regenerate after birth. Supporting cells, whichsurround hair cells in the normal cochlear epithelium, have potential todifferentiate into new hair cells in the neonatal mouse followingototoxic damage (Bramhall et al. 2014). Using lineage tracing, the newhair cells, predominantly outer hair cells, have been shown to arisefrom Lgr5-expressing inner pillar and third Deiters cells, and new haircell generation has been shown to incrementally be increased bypharmacological inhibition of Notch (Bramhall et al. 2014, Mizutari etal. 2014). It has been postulated that the neonatal mammalian cochleahas some capacity for hair cell regeneration following damage alone (Coxet al. 2014) and that Lgr5-positive (Lgr5⁺) cells act as hair cellprogenitors in the cochlea (Chai et al. 2011, Shi et al. 2012).

Auditory dysfunction in humans is an ongoing problem in the medicalfields of otology and audiology. Auditory dysfunctions typically arisefrom both acute and chronic exposures to loud sounds, ototoxicchemicals, and aging. Sounds exceeding 85 decibels can cause hearingloss and is generated by sound sources such as, gun shots, explodingbombs, jet engines, power tools, and musical concerts. Other commoneveryday activities and products also give rise to high intensity noisesuch as use of hair dryers, MP3 players, lawn mowers, and blenders.Military personnel are particularly at risk for noise induced hearingloss due to typical military noise exposures. Side effects ofnoise-induced hearing loss include tinnitus (ringing in the ears),diminished speech understanding, hyperacusis, recruitment and varioustypes of auditory processing impairments. Exposures to commonly usedmedications may also induce auditory dysfunctions. For instance,patients treated with anticancer therapies, antibiotics and othermedications often develop hearing loss as a side effect. Furthermore,exposure to industrial chemicals and gasses may induce auditoryimpairments. Auditory dysfunction is a common consequence of aging inWestern societies. Hearing impairments can be attributed to a widevariety of causes, including infections (e.g., otitis media), geneticpredisposition, mechanical injury, tumors, loud sounds or prolongedexposure to noise, aging, and chemical-induced ototoxicity (e.g.,antibiotics or platin drugs) that damages neurons and/or hair cells ofthe peripheral auditory system. This can be caused by acute noise or canbe progressive over time.

Currently, very few cases of hearing loss can actually be cured.Audiological devices such as hearing aids have limitations including theinability to improve speech intelligibility. Of those impacted byhearing impairments, less than 20 percent presently use hearinginstruments. In cases of age-related, noise- or drug-induced auditorydysfunctions, often the only effective way to currently “treat” thedisorder or reduce its severity is prevention: avoiding excessive noiseand using ear protectors, practicing a healthy lifestyle, and avoidingexposure to ototoxic drugs and substances if possible.

Once the hearing loss has developed, people may use a hearing aid tocorrect the hearing loss. However, despite advances in the performanceof these prostheses, they still have significant limitations. Forexample, hearing aids mainly amplify sound and cannot correct forsuprathreshold or retrocochlear impairments such as impaired speechintelligibility, speech in noise deficits, tinnitus, hyperacusis,loudness recruitment and various other types of central auditoryprocessing disorders. Hearing aids essentially amplify sounds, whichstimulate unimpaired cells, but there is no therapy for aiding recoveryof impaired cells or maximizing the function of existing unimpairedcells.

In cases of complete or profound deafness, a cochlear implant may beused. This device transmits electrical stimuli via electrodes surgicallyimplanted into the cochlea. A cochlear implant can be of particular helpfor deaf children if it is implanted around the age of two or three, thetime when language skills are developing fastest. However, cochlearimplants involve invasive surgery and are expensive. Furthermore,cochlear implants require viable neurons to achieve benefit.

Approximately 17 percent of Americans have hearing loss and half of thatnumber are under the age of 65. It is predicted that the number ofAmericans with hearing loss will exceed 70 million by the year 2030.

About 300 million people worldwide currently suffer from moderate tosevere hearing loss, and this number is expected to increase to 700million by the year 2015. Most of these people will suffer from noiseinduced hearing loss and one in four Americans will develop permanenthearing loss as a result of occupational exposure to noise hazards.According to the Center for Commercialization of Advanced Technology,the Department of Defense and the VA, the VA spends over $1 billion onhearing loss compensation. The Navy, Marine Corps, and Air Force(combined) file 22,000 new hearing loss claims, and hearing loss coststhe economy more than $56 billion per year.

Thus, there remains a long felt need to protect auditory cells beforeinjury and preserve/promote the function of existing cells after injury.As disclosed below, in certain embodiments, the present inventionprovides compositions, systems, and methods for preventing and treatingauditory dysfunctions.

There are many patient populations that could be helped with newtherapies that prevent or treat hearing loss, for example, patients withvertigo, tinnitus, or patients who require a cochlear implant, those whohave hearing loss but are not eligible for a cochlear implants, andthose with chronic mild/moderate or severe hearing loss.

Previous work has shown that manipulating direct inhibitors of cycleactivation (e.g., p27kip1, Rb1, p19ink4d, p21cip1) causes many cells,including hair cells, to proliferate. Hair cells that re-enter cellcycle subsequently die and hearing ability deteriorates (Salvi R. J.Hair Cell Regeneration Repair and Protection, Sage et al 2005, 2006).Differentiation to hair cells was not seen after supporting cellproliferation resulting from manipulation of cell cycle genes, such asp27^(Kip1) or Rb (Yu et al. 2010, Liu et al. 2012).

Stem cells exhibit an extraordinary ability to generate multiple celltypes in the body. Besides embryonic stem cells, tissue specific stemcells serve a critical role during development as well as in homeostasisand injury repair in the adult. Stem cells renew themselves throughproliferation as well as generate tissue specific cell types throughdifferentiation. The characteristics of different stem cells varies fromtissue to tissue, and are determined by their intrinsic genetic andepigenetic status. However, the balance between self-renewal anddifferentiation of different stem cells are all stringently controlled.Uncontrolled self-renewal may lead to overgrowth of stem cells andpossibly tumor formation, while uncontrolled differentiation may exhaustthe stem cell pool, leading to an impaired ability to sustain tissuehomeostasis. Thus, stem cells continuously sense their environment andappropriately respond with proliferation, differentiation or apoptosis.It would be desirable to drive regeneration by controlling the timingand extent of stem cell proliferation and differentiation. Controllingthe proliferation with small molecules that are cleared over time wouldallow for control of the timing and extent of stem cell proliferationand differentiation. Remarkably, tissue stem cells from differenttissues share a limited number of signaling pathways for the regulationof their self-renewal and differentiation, albeit in a very contextdependent manner. One of these pathways is the Notch pathway.

The Notch pathway represents an evolutionarily conserved signalingpathway that possesses a simple but unique mode of action. The coreNotch pathway contains only a small number of components. The canonicalNotch pathway is activated through the binding of Notch ligand on thesurface of signal-sending cells to the Notch receptor on neighborsignal-receiving cells. This event initiates a cascade of proteolyticcleavages of the Notch receptor, including γ-secretase-mediated releaseof the Notch Intracellular Domain (NICD). NICD fragment then enters thenucleus to induce target gene transcription. Under most circumstances,the canonical Notch pathway requires physical contact betweenneighboring cells, thus it links the fate of one cell to that of animmediate neighbor, providing a sophisticated way to control theself-renewal and differentiation of stem cells. The Notch pathway hasbeen shown to regulate many types of stem cells, including embryonicstem cells, neural stem cells, hematopoietic stem cells as well as Lgr5epithelial stem cells (Koch et al, 2013; VanDussen et al, 2012).

Lgr5 is expressed across a diverse range of tissues and has beenidentified as a biomarker of adult stem cells in certain tissues such asthe gut epithelia (Barker et al. 2007), kidney, hair follicle, andstomach (Barker et al, 2010; Haegebarth & Clevers, 2009). It was firstpublished in 2011, that mammalian inner ear hair cells are derived fromLGR5⁺ cells (Chai et al, 2011, Shi et al. 2012). Lgr5 is a knowncomponent of the Wnt/beta-catenin pathway, which has been shown to playmajor roles in differentiation, proliferation, and inducing stem cellcharacteristics (Barker et al. 2007).

Prior work has focused on transdifferentiation of supporting cells intohair cells through activation or forced expression of genes that lead tohair cell formation, with a particular focus on mechanisms to enhanceexpression of Atoh1 (Bermingham et al., 1999; Zheng and Gao, 2000;Izumikawa et al., 2005; Mizutari et al., 2013). Interestingly, cellstransduced with Atoh1 vectors have been shown to acquire vestibularphenotypes (Kawamoto et al., 2003; Huang et al., 2009; Yang et al.,2012, 2013), and lack complete development. As mentioned, upregulatingAtoh1 via gene insertion has been shown to create non-cochlear celltypes that behave in a manner that is not found within the nativecochlea. In addition, these methods increase hair cell numbers butdecrease supporting cell numbers. Since supporting cells are known tohave specialized roles (Ramirez-Camancho 2006, Dale and Jagger 2010),loss of these cells could create problems in proper cochlear function.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure, therefore, may benoted a method for activating the Wnt pathway in a cochlear supportingcell population to increase the capacity of the population forself-renewal, i.e., the capacity for repeated generation of daughtercells with equivalent proliferation and ‘cell fate specification’potential, and differentiation, i.e., the capacity for generation ofdaughter cells specified for differentiation. Preferably, the Wntpathway is activated upstream of the c-myc gene in members of thepopulation and without any genetic modification of the population.Instead, the Wnt pathway is preferably activated by small molecules thattransiently induce such activity. Additionally, the supporting cellpopulation preferably includes supporting cells that are LGR5⁺ andendogenous to the Organ of Corti.

A further aspect of the present disclosure is a method for inducing theself-renewal of stem/progenitor supporting cells comprised by a cochlearcell population. That is, the stem/progenitor supporting cells areinduced to proliferate (i.e., divide and form daughter cells) whilemaintaining, in the daughter cells, the capacity to differentiate intohair cells. In contrast, if the stem/progenitor supporting cells weremerely induced to proliferate (without maintaining multi-potency), thedaughter cells would lack the capacity to divide into hair cells.Further, merely enforcing differentiation of a pre-existingstem/progenitor cell population has the potential to exhaust the stemcell pool. Accordingly, the present disclosure provides a method inwhich pre-existing cochlear supporting cells are induced to proliferateprior to differentiation and the expanded population is then permitted(or even induced in some embodiments) to differentiate into hair cells.Preferably, proliferation is induced by activating the Wnt pathwayupstream of the c-myc gene in members of the population and without anygenetic modification of the population. Instead, proliferation ispreferably activated by small molecules that transiently induce suchactivity. Additionally, in certain embodiments the supporting cellpopulation preferably includes supporting cells that are LGR5⁺ andendogenous to the Organ of Corti.

In certain embodiments, therefore, the present disclosure providescompositions that have the capacity to induce self-renewal of apopulation of supporting cells. These compositions have the capacity toactivate pathways and mechanisms that are known to be involved ininducing stem cell properties, such as those used to create “inducedpluripotent stem cells” (combined Wnt stimulation, HDAC inhibition,TGF-beta inhibition, RAR activation, DKK1 suppression). Preferably, thepathways are activated with small molecules. For example, a preferredcomposition when applied in vitro to a supporting cell populationinduces the population to proliferate to a high degree and in highpurity in a Stem Cell Proliferation Assay, and also allows thepopulation to differentiate into a high purity population of hair cellsin a Stem Cell Differentiation Assay. In one such embodiment, thecomposition induces and maintains stem cell properties by proliferatingto produce stem cell that can divide for many generations and maintainthe ability to have a high proportion of the resulting cellsdifferentiate into hair cells. Further, the proliferating stem cellsexpress stem cell markers which may include one or more of Lgr5, Sox2,Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3,Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tcl1,Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1,Smad2, smad2/3, smad4, smad5, and smad7.

In certain embodiments, the disclosure provides a method for expanding apopulation of cochlear cells in a cochlear tissue comprising a parentpopulation of cells. In this embodiment, the method comprises contactingthe cochlear tissue with a stem cell proliferator to form an expandedpopulation of cells in the cochlear tissue, wherein

-   -   the stem cell proliferator is capable of (i) forming a        proliferation assay final cell population from a proliferation        assay initial cell population over a proliferation assay time        period in a stem cell proliferation assay and (ii) forming a        differentiation assay final cell population from a        differentiation assay initial cell population over a        differentiation assay time period in a stem cell differentiation        assay wherein:    -   (a) the proliferation assay initial cell population has (i) a        proliferation assay initial number of total cells, (ii) a        proliferation assay initial number of Lgr5⁺ cells, (iii) a        proliferation assay initial number of hair cells, (iv) a        proliferation assay initial Lgr5⁺ cell fraction that equals the        ratio of the proliferation assay initial number of Lgr5⁺ cells        to the proliferation assay initial number of total cells,        and (v) a proliferation assay initial hair cell fraction that        equals the ratio of the proliferation assay initial number of        hair cells to the proliferation assay initial number of total        cells;    -   (b) the proliferation assay final cell population has (i) a        proliferation assay final number of total cells, (ii) a        proliferation assay final number of Lgr5⁺ cells, (iii) a        proliferation assay final number of hair cells, (iv) a        proliferation assay final Lgr5⁺ cell fraction that equals the        ratio of the proliferation assay final number of Lgr5⁺ cells to        the proliferation assay final number of total cells and (v) a        proliferation assay final hair cell fraction that equals the        ratio of the proliferation assay final number of hair cells to        the proliferation assay final number of total cells;    -   (c) the differentiation assay initial cell population has (i) a        differentiation assay initial number of total cells, (ii) a        differentiation assay initial number of Lgr5⁺ cells, (iii) a        differentiation assay initial number of hair cells, (iv) a        differentiation assay initial Lgr5 cell fraction that equals the        ratio of the differentiation assay initial number of Lgr5⁺ cells        to the differentiation assay initial number of total cells,        and (v) a differentiation assay initial hair cell fraction that        equals the ratio of the differentiation assay initial number of        hair cells to the differentiation assay initial number of total        cells;    -   (d) the differentiation assay final cell population has (i) a        differentiation assay final number of total cells, (ii) a        differentiation assay final number of Lgr5⁺ cells, (iii) a        differentiation assay final number of hair cells, (iv) a        differentiation assay final Lgr5⁺ cell fraction that equals the        ratio of the differentiation assay final number of Lgr5⁺ cells        to the differentiation assay final number of total cells,        and (v) a differentiation assay final hair cell fraction that        equals the ratio of the differentiation assay final number of        hair cells to the differentiation assay final number of total        cells;    -   (e) the proliferation assay final number of Lgr5⁺ cells exceeds        the proliferation assay initial number of Lgr5⁺ cells by a        factor of at least 10; and    -   (f) the differentiation assay final number of hair cells is a        non-zero number.

In certain embodiments, the stem cell proliferator comprises StemnessDriver. In certain embodiments, the stem cell proliferator comprises aDifferentiation Inhibitor. In certain embodiments, the stem cellproliferator comprises a Stemness Driver and a DifferentiationInhibitor.

In certain embodiments, the disclosure provides a method for increasingthe cell density of supporting cells in a population of cochlear cells.The method comprises activating pathways and mechanisms that induce stemcell properties in the supporting cells, proliferating the activatedsupporting cells (while maintaining the multi-potent character of thesupporting cells in the newly formed daughter cells) and thereafterallowing (or even inducing) the expanded population to differentiateinto hair cells to form an expanded cochlear cell population wherein thecell density of hair cells in the expanded cochlear cell populationexceeds the cell density of hair cells in the original (non-expanded)cochlear cell population. In some embodiments, the supporting cellpopulation is an in vitro supporting cell population. In otherembodiments, the supporting cell population is an in vivo supportingcell population. Additionally, the proliferation stage is preferablycontrolled to substantially maintain the native organization of thecochlear structure. Preferably, proliferation is induced by smallmolecules that transiently induce such activity rather than by inductionof c-myc and without any genetic modification of the population.Additionally, in certain embodiments the supporting cell populationpreferably includes supporting cells that are LGR5′ and endogenous tothe Organ of Corti.

In certain embodiments, the disclosure provides a method for increasingthe cell density of Lgr5⁺ supporting cells in a population of cochlearcells. The method comprises activating pathways and mechanisms thatinduce or maintain stem cell properties in the Lgr5+ supporting cells,proliferating the activated Lgr5⁺ supporting cells (while maintainingsuch stem cell properties) and thereafter allowing (or even inducing)the expanded population to differentiate into hair cells to form anexpanded cochlear cell population wherein the cell density of hair cellsin the expanded cochlear cell population exceeds the cell density ofhair cells in the original (non-expanded) cochlear cell population. Insome embodiments, the Lgr5⁺ supporting cell population is an in vitroLgr5⁺ stem cell population. In other embodiments, the Lgr5+ supportingcell population is an in vivo supporting cell population. Additionally,in certain embodiments the proliferation stage is preferably controlledto substantially maintain the native organization of the cochlearstructure.

In certain embodiments, a composition containing a Stemness Driver and aDifferentiation Inhibitor is administered to a cochlear cell populationto induce proliferation of stem cells and to inhibit differentiation ofthe stem cells until the desired expansion of the stem cell populationis achieved. Thereafter, the expanded population is permitted (oroptionally even induced) to differentiate into hair cells. Additionally,the proliferation stage is preferably controlled to substantiallymaintain the native organization of the cochlear structure. In someembodiments, the Sternness Driver and Differentiation inhibitor aresmall molecules. In some embodiments, the stem cell population is an invivo stem cell population. In other embodiments, the stem cellpopulation is an in vitro stem cell population. In some embodiments, thestem cell population is an in vivo Lgr5⁺ stem cell population. In otherembodiments, the stem cell population is an in vitro Lgr5⁺ stem cellpopulation.

In certain embodiments, the disclosure provides a method for increasingthe cell density of hair cells in an initial population of cochlearcells, the initial population (which may be an in vivo or an in vitropopulation) comprises hair cells, Lgr⁻ supporting cells, and Lgr5⁺supporting cells. The method comprises administering to the initialpopulation a composition that contains a Stemness Driver and aDifferentiation Inhibitor wherein the composition has the capacity toinduce the expansion of the number of Lgr5⁺ supporting cells in thepopulation in a Stem Cell Proliferation Assay, and allows Lgr5⁺supporting cells within the population to differentiate into apopulation of hair cells in a Stem Cell Differentiation Assay.

In certain embodiments, the method produces stem cells in a Stem CellProliferation Assay that express stem cells markers Lgr5⁺. In certainembodiments, if a mixed population of Lgr5⁺ and non-Lgr5⁺ stems areplaced in a Stem Cell Proliferation Assay, the method increases thefraction of cells in the population that are Lgr5⁺.

Expanding supporting cell populations to a degree that destroys thenative organization of the cochlear structure could inhibit cochlearfunction. Driving proliferation of existing supporting cells with asmall molecule signal may allow for a more controlled regeneration ofhair cells than using gene delivery, which is incapable of targeting aspecific cell type and permanently alters a cell's genetic information.An approximately normal cochlear structure is desired with rows of haircells that have supporting cells between them, and hair cells do notcontact other hair cells. Further, it would be desirable to avoid usinggenetic modification to drive proliferation to create large cellaggregations in the cochlea that disrupt the organ's anatomy. In certainembodiments, it may be preferable to use a composition that isnon-oncogenic. In certain embodiments, it may be preferable to use acomposition that proliferates stem cells independent of the operation ofthe c-myc pathway, for instance in a mechanism which is effective in ac-myc knock-out or where c-myc is inhibited or silenced.

In certain embodiments, the disclosure provides a composition comprisinga Stemness Driver that may be used to drive the selective expansion ofcochlea supporting cells. In some cases, a Sternness Driver may alsoinduce differentiation of the supporting cells to hair cells if aDifferentiation Inhibitor is not present at an Effective DifferentiationInhibition Concentration. Examples of Stemness Drivers that may driveboth proliferation and differentiation include GSK3Beta inhibitors andWnt agonists. In certain embodiments, the proliferation of the stemcells may be enhanced by adding a modulator of cell cycle regulators,such as the p27 or TgfBeta pathways. In certain of these embodiments,the composition comprises the Stemness Driver and DifferentiationInhibitor in a formulation that releases the Stemness Driver andDifferentiation inhibitor at different rates in a Release Assay. Thus,for example, in one such embodiment the formulation may provide aconstant, sustained, extended, delayed or pulsatile rate of release ofthe an active agent into the inner ear environment and thus avoid anyvariability in drug exposure.

In some embodiments, a Stemness Driver may be used to drive theproliferation of Lgr5⁺ stem cells. In some cases, a Stemness Driver mayalso induce differentiation of LGR5+ cells to hair cells if aDifferentiation Inhibitor is not present at an Effective DifferentiationInhibition Concentration. Examples of Stemness Drivers that may driveboth proliferation and differentiation include GSK3Beta inhibitors andWnt agonists. In certain of these embodiments, the composition comprisesthe Stemness Driver and a Differentiation Inhibitor in a formulationthat releases the Stemness Driver and Differentiation Inhibitor atdifferent rates in a Release Assay. Thus, for example, in one suchembodiment the formulation may provide a constant, sustained, extended,delayed or pulsatile rate of release of the an active agent into theinner ear environment and thus avoid any variability in drug exposure.

In certain embodiments, the disclosure provides a method for increasingthe cell density of hair cells in an initial population of cochlearcells comprising hair cells and supporting cells. The method comprisesselectively expanding the number of supporting cells in the initialpopulation to form an intermediate cochlear cell population wherein theratio of the number of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of the number ofsupporting cells to hair cells in the initial cochlear cell population.The method further comprises generating hair cells in the intermediatecochlear cell population to form an expanded cochlear cell populationwherein the ratio of the number of hair cells to supporting cells in theexpanded cochlear cell population exceeds the ratio of the number ofhair cells to supporting cells in the intermediate cochlear cellpopulation.

In certain embodiments, the disclosure provides a method for increasingthe number of Lgr5⁺ supporting cells or increasing the Lgr5⁺ activity inan initial population of cochlear cells, wherein the initial populationcomprises supporting cells and hair cells. For example, in one suchmethod an intermediate population is formed in which the number of Lgr5⁺supporting cells is expanded relative to the initial population.Alternatively, in one such method an intermediate population is formedin which the Lgr5 activity of the supporting cells relative to theinitial population is increased. Alternatively, a method where thenumber of Lgr5⁺ cells is increased relative to the initial cellpopulation by activating Lgr5⁺ expression in cell types that normallylack or have very low levels of Lgr5. By way of further example, anintermediate population is formed in which the number of Lgr5⁺supporting cells is expanded and the Lgr5⁺ activity is increasedrelative to the initial cochlear cell population. Thereafter, hair cellsin the intermediate cochlear cell population may be generated to form anexpanded cochlear cell population wherein the ratio of hair cells tosupporting cells in the expanded cochlear cell population exceeds theratio of the number of hair cells to supporting cells in theintermediate cochlear cell population.

In some embodiments, the method applied to an adult mammal produces apopulation of adult mammalian Lgr5⁺ cells that are in S-phase.

In each of the aforementioned embodiments of the present disclosure, theDifferentiation Inhibitor may be a Notch agonist or HDAC inhibitor. Insome such embodiments, there may be a first Proliferation Period with anEffective Stemness Driver Concentration and an Effective DifferentiationInhibition Concentration of a Differentiation Inhibitor, followed by aDifferentiation Period with an Effective Stemness Driver Concentrationand without an Effective Differentiation Inhibition Concentration of aDifferentiation Inhibitor. In each of these embodiments, the StemnessDriver and Differentiation Inhibitor are provided to the cochlear cellsin a formulation that releases the Stemness Driver and Differentiationinhibitor at different rates. For example, the formulation may provide aconstant, sustained, extended, delayed or pulsatile rate of release ofthe Stemness Driver and the Differentiation Inhibitor into the inner earenvironment. Significantly, however, the formulation releases theStemness Driver and Differentiation Inhibitor in a manner to provide afirst Proliferation Period with an Effective Stemness DriverConcentration and an Effective Differentiation Inhibition Concentrationof the Differentiation Inhibitor, followed by a Differentiation Periodwith an Effective Stemness Driver Concentration and without an EffectiveDifferentiation Inhibition Concentration of the DifferentiationInhibitor.

In certain embodiments, the method further comprises performing highthroughput screening on inner ear progenitor/stem cells to identifyStemness Drivers and/or Differentiation Inhibitors, as used in a StemCell Proliferation Assay or Stem Cell Differentiation Assay.

In each of the aforementioned embodiments of the present disclosure,there may be a first Proliferation Period with an Effective StemnessDriver Concentration of a Wnt agonist or GSK3Beta inhibitor and anEffective Differentiation Inhibition Concentration of a Notch agonist orHDAC inhibitor, followed by a Differentiation Period with an EffectiveStemness Driver Concentration of a Wnt agonist or GSK3Beta inhibitor andwithout an Effective Differentiation Inhibition Concentration of a Notchagonist or HDAC inhibitor. In each of these embodiments, therefore, theformulation may provide a constant, sustained, extended, delayed orpulsatile rate of release of the Wnt agonist or GSK3Beta inhibitor andthe Notch agonist or HDAC inhibitor into the inner ear environment.Significantly, however, the formulation releases the Wnt agonist orGSK3Beta inhibitor and the Notch agonist or HDAC inhibitor in a mannerto provide a first Proliferation Period with an Effective StemnessDriver Concentration of the Wnt agonist or GSK3Beta inhibitor and anEffective Differentiation Inhibition Concentration of the Notch agonistor HDAC inhibitor, followed by a Differentiation Period with anEffective Stemness Driver Concentration of the Wnt agonist or GSK3Betainhibitor and without an Effective Differentiation InhibitionConcentration of the Notch agonist or HDAC inhibitor.

In some embodiments, the Differentiation inhibitor is also a StemnessDriver. In some embodiments, the Differentiation inhibitor is a Notchagonist and is also a Stemness Driver. In some embodiments, theDifferentiation inhibitor is Valproic Acid, which may be a StemnessDriver. If a Differentiation Inhibitor is also a Stemness Driver, theconcentration of the Differentiation Inhibitor should fall below theEffective Differentiation Inhibition Concentration during theDifferentiation Period.

In each of the aforementioned embodiments, the Differentiation inhibitorand Stemness Driver may be contained within a sustained release polymergel. In certain embodiments, the gel may be injected through a needlebut becomes solid in the middle ear space. In certain embodiments, thegel is comprised of a thermoreversible polymer such as Poloxamer 407.

The Notch pathway is known to be a key regulator of the differentiationprocess from supporting cells to hair cells (Lanford et al. 1999). Insome embodiments, a Stemness Driver and Differentiation Inhibitor areapplied such that the Notch activity level in supporting cells remainsat 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the Notch activitylevel in supporting cells in the native state.

In certain embodiments, the disclosure provides a method of andcompositions for generating hair cells, the method comprising:administering or causing to be administered to a stem cell population(e.g., of an in vitro, ex vivo, or in vivo sample/subject) a compositioncomprising both of (i) and (ii): (i) a GSK3-beta inhibitor (or aderivative or pharmaceutically acceptable salt thereof) and/or Wntagonist (or a derivative or pharmaceutically acceptable salt thereof),and (ii) a Notch agonist (or a derivative or pharmaceutically acceptablesalt thereof) and/or HDAC inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof), thereby proliferating stem cells in the stemcell population and resulting in an expanded population of stem cells;and exposing the expanded population of stem cells to a GSK3-betainhibitor (or a derivative or pharmaceutically acceptable salt thereof)and/or a Wnt agonist (or a derivative or pharmaceutically acceptablesalt thereof), and, optionally, a notch inhibitor (or a derivative orpharmaceutically acceptable salt thereof), thereby facilitatinggeneration of inner ear hair cells from the expanded population of stemcells.

In certain embodiments, the disclosure provides compositions, systems,and methods for preventing and treating auditory dysfunction. Forexample, in certain embodiments, the disclosure provides methods forpreventing or treating auditory impairments in a subject comprisingadministering to said subject an effective amount of a compositioncomprising (a) (i) an HDAC inhibitor and/or Notch activator and (ii) aGSK3-beta inhibitor, a derivative thereof, e.g., a derivative of an HDACinhibitor, a derivative of a Notch activator, and/or a derivative of aGSK3-beta inhibitor, a pharmaceutically acceptable salt thereof, [e.g.,a pharmaceutically acceptable salt of an HDAC inhibitor, apharmaceutically acceptable salt of a Notch activator, and/or apharmaceutically acceptable salt of a GSK3-beta inhibitor, or acombination thereof and (b) a pharmaceutically acceptable carrier orexcipient, so as to treat auditory impairments in the subject. Thus, forexample, the composition may comprise (a) an HDAC inhibitor (or aderivative or pharmaceutically acceptable salt thereof) and a GSK3-betainhibitor (or a derivative or pharmaceutically acceptable salt thereof),and (b) a pharmaceutically acceptable carrier or excipient. By way offurther example, the composition may comprise (a) a Notch activator (ora derivative or pharmaceutically acceptable salt thereof) and aGSK3-beta inhibitor (or a derivative or pharmaceutically acceptable saltthereof), and (b) a pharmaceutically acceptable carrier or excipient. Byway of further example, the composition may comprise (a) an HDACinhibitor (or a derivative or pharmaceutically acceptable salt thereof),a Notch activator (or a derivative or pharmaceutically acceptable saltthereof), and a GSK3-beta inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof), and (b) a pharmaceutically acceptable carrieror excipient.

In certain embodiments, the present disclosure also relates to ex-vivouses of cells described herein. For example, approaches described hereincan be used for hi and for discovery purposes. For example, certainembodiments of the present disclosure are useful for identifying agentsthat proliferate hair cell progenitors and/or increase numbers of haircells, and also agents that protect supporting cells and/or hair cells(e.g. to support their survival), and also for identifying agents thatare toxic or not toxic to supporting cells or differentiated progenyincluding hair cells.

In certain embodiments, the disclosure provides methods for preventingor treating auditory impairments in a subject in need of treatmentcomprising administering to said subject an effective amount of acomposition comprising, an HDAC inhibitor and/or Notch activator and aGSK3beta inhibitor or derivative thereof or pharmaceutically acceptablesalt thereof and an acceptable carrier or excipient, so as to treatauditory impairments in the subject.

In certain embodiments, the disclosure provides for methods forinhibiting the loss or death of the cells of the auditory system in asubject comprising administering to said subject an effective amount ofa composition described herein or derivative thereof or pharmaceuticallyacceptable salt thereof and an acceptable carrier or excipient, therebyinhibiting loss or death of the cells of the auditory system in thesubject.

In certain embodiments, the disclosure provides methods for maintainingor promoting the growth of cells of the auditory system in a subjectcomprising administering to said subject a composition comprising as anagent described herein or derivative thereof or pharmaceuticallyacceptable salt thereof and an acceptable carrier or excipient in aneffective amount so as to augment or initiate endogenous repair, therebymaintaining or promoting the growth of cells of the auditory system inthe subject.

The methods and compositions of the present disclosure allow greater andthus more effective dosing with these ototoxicity-inducingpharmaceutical drugs, while concomitantly preventing or reducingototoxic effects caused by these drugs. The methods of the presentdisclosure provide a safe, effective, and prolonged means forprophylactic or curative treatment of hearing impairments related toinner ear tissue damage, loss, or degeneration, particularly sound oraging-induced, and ototoxin-induced, and particularly involving innerear hair cells. In certain embodiments, the present disclosure providescompositions and methods that address one or more of these or othergoals.

This disclosure generally relates to compositions, systems, and methodsfor inducing, promoting, or enhancing the growth, proliferation, orregeneration of inner ear tissue, for example, inner ear supportingcells and/or inner ear hair cells.

Also described herein is a method for expanding a population of cochlearcells in a cochlear tissue comprising a parent population of cells, theparent population including supporting cells and a number of Lgr5⁺cells, the method comprising contacting the cochlear tissue with a stemcell proliferator to form an expanded population of cells in thecochlear tissue, wherein the stem cell proliferator is capable (i) in astem cell proliferation assay of increasing the number of Lgr5⁺ cells ina stem cell proliferation assay cell population by a factor of at least10 and (ii) in a stem cell differentiation assay of forming hair cellsfrom a cell population comprising Lgr5⁺ cells.

Also described herein is a method for expanding a population of cochlearcells in a cochlear tissue comprising a parent population of cells, theparent population including supporting cells, the method comprisingcontacting the cochlear tissue with a stem cell proliferator to form anexpanded population of cells in the cochlear tissue. The stem cellproliferator can be capable of (i) forming a proliferation assay finalcell population from a proliferation assay initial cell population overa proliferation assay time period in a stem cell proliferation assay and(ii) forming a differentiation assay final cell population from adifferentiation assay initial cell population over a differentiationassay time period in a stem cell differentiation assay wherein: (a) theproliferation assay initial cell population has (i) a proliferationassay initial number of total cells, (ii) a proliferation assay initialnumber of Lgr5⁺ cells, (iii) a proliferation assay initial number ofhair cells, (iv) a proliferation assay initial Lgr5⁺ cell fraction thatequals the ratio of the proliferation assay initial number of Lgr5⁺cells to the proliferation assay initial number of total cells, and (v)a proliferation assay initial hair cell fraction that equals the ratioof the proliferation assay initial number of hair cells to theproliferation assay initial number of total cells; (b) the proliferationassay final cell population has (i) a proliferation assay final numberof total cells, (ii) a proliferation assay final number of Lgr5⁺ cells,(iii) a proliferation assay final number of hair cells, (iv) aproliferation assay final Lgr5⁺ cell fraction that equals the ratio ofthe proliferation assay final number of Lgr5⁺ cells to the proliferationassay final number of total cells and (v) a proliferation assay finalhair cell fraction that equals the ratio of the proliferation assayfinal number of hair cells to the proliferation assay final number oftotal cells; (c) the differentiation assay initial cell population has(i) a differentiation assay initial number of total cells, (ii) adifferentiation assay initial number of Lgr5⁺ cells, (iii) adifferentiation assay initial number of hair cells, (iv) adifferentiation assay initial Lgr5⁺ cell fraction that equals the ratioof the differentiation assay initial number of Lgr5⁺ cells to thedifferentiation assay initial number of total cells, and (v) adifferentiation assay initial hair cell fraction that equals the ratioof the differentiation assay initial number of hair cells to thedifferentiation assay initial number of total cells; (d) thedifferentiation assay final cell population has (i) a differentiationassay final number of total cells, (ii) a differentiation assay finalnumber of Lgr5⁺ cells, (iii) a differentiation assay final number ofhair cells, (iv) a differentiation assay final Lgr5⁺ cell fraction thatequals the ratio of the differentiation assay final number of Lgr5⁺cells to the differentiation assay final number of total cells, and (v)a differentiation assay final hair cell fraction that equals the ratioof the differentiation assay final number of hair cells to thedifferentiation assay final number of total cells; (e) the proliferationassay final number of Lgr5⁺ cells exceeds the proliferation assayinitial number of Lgr5⁺ cells by a factor of at least 10; and (f) thedifferentiation assay final number of hair cells is a non-zero number.

The proliferation assay final number of Lgr5⁺ cells can be greater thanthe proliferation assay initial number of Lgr5⁺ cells by a factor of atleast 50, or by a factor of at least 100. The expanded population ofcells in the cochlear tissue can include a greater number of hair cellsthan does the parent population. The proliferation assay final Lgr5⁺cell fraction can be greater than the differentiation assay initialLgr5⁺ cell fraction by at least a factor of 2. The differentiation assayfinal hair cell fraction can be greater than the proliferation assayinitial hair cell fraction by at least a factor of 2. The proliferationassay final hair cell fraction can be at least 25% less than theproliferation assay initial hair cell fraction. The proliferation assayfinal Lgr5⁺ cell fraction can be at least 10% greater than proliferationassay initial Lgr5⁺ cell fraction. One of more morphologicalcharacteristics of the cochlear tissue can be maintained. Nativemorphology can be maintained. The at least one stem cell proliferatorcan be dispersed in a biocompatible matrix, which can be a biocompatiblegel or foam. The composition can be a controlled release formulation.The cochlear tissue can be an in vivo cochlear tissue or an ex vivocochlear tissue. The method can produce a population of Lgr5⁺ cells thatare in s-phase. The at least one stem cell proliferator can include botha stemness driver and a differentiation inhibitor. Contacting canprovide to the cochlear tissue: in an initial phase, at least aneffective proliferation concentration of the stemness driver and atleast an effective differentiation inhibition concentration of thedifferentiation inhibitor; and in a subsequent phase, at least aneffective proliferation concentration of the stemness driver and lessthan an effective differentiation inhibition concentration of thedifferentiation inhibitor. The cochlear tissue can be in a subject, andcontacting the cochlear tissue with the composition can be achieved byadministering the composition trans-tympanically to the subject.Contacting the cochlear tissue with the composition can result inimproved auditory functioning of the subject.

Also described herein is a composition that includes a biocompatiblematrix and at least one stem cell proliferator, wherein the at least onestem cell proliferator is capable, in a stem cell proliferation assay,of expanding an initial test population of Lgr5⁺ cells to create anexpanded test population, and wherein the expanded test population hasat least 10-fold more Lgr5⁺ cells than does the initial test population.

Also described herein is a composition that includes a biocompatiblematrix and at least one stem cell proliferator, wherein the at least onestem cell proliferator is capable, in a stem cell proliferation assay,of expanding an initial cell population containing Lgr5⁺ cells to createa final cell population, and wherein the final cell population has atleast 10-fold more Lgr5⁺ cells than does the initial cell population.Additionally, (a) the initial cell population has (i) an initial numberof total cells, (ii) an initial number of Lgr5⁺ cells, (iii) an initialnumber of hair cells, (iv) an initial Lgr5⁺ cell fraction that equalsthe ratio of the proliferation assay initial number of Lgr5⁺ cells tothe proliferation assay initial number of total cells, and (v) aninitial hair cell fraction that equals the ratio of the initial numberof hair cells to initial number of total cells; and (b) the final cellpopulation has (i) a final number of total cells, (ii) a final number ofLgr5 cells, (iii) a final number of hair cells, (iv) a final Lgr5⁺ cellfraction that equals the ratio of the final number of Lgr5⁺ cells to thefinal number of total cells and (v) a final hair cell fraction thatequals the ratio of the final number of hair cells to the final numberof total cells.

The final number of Lgr5⁺ cells can be greater than the initial numberof Lgr5⁺ cells by a factor of at least 50, or by a factor of at least100.

The at least one stem cell proliferator can be dispersed in abiocompatible matrix, which can be a biocompatible gel or foam. Theproliferation assay final Lgr5⁺ cell fraction can be at least 10%greater than the proliferation assay initial Lgr5⁺ cell fraction. The atleast one stem cell proliferator can include at least one of a stemnessdriver and a differentiation inhibitor. The at least one stem cellproliferator can include both a stemness driver and a differentiationinhibitor. The stem cell proliferator can include a stemness driver in aconcentration that is 100-fold greater than an effective proliferationconcentration of the stemness driver and a differentiation inhibitor ina concentration that is at least 100-fold greater than an effectivedifferentiation inhibition concentration of the differentiationinhibitor. The composition can be a controlled release formulation. Thecontrolled release formulation when administered to a subjecttrans-tympanically imparts an immediate release, a delayed release, asustained release, an extended release, a variable release, a pulsatilerelease, or a bi-modal release of the stem cell proliferator. Thecontrolled release formulation when administered to a subject canprovide: (a) in an initial phase, at least an effective proliferationconcentration of the stemness driver and at least an effectivedifferentiation inhibition concentration of the differentiationinhibitor; and (b) in a subsequent phase, at least an effectiveproliferation concentration of the stemness driver and less than aneffective differentiation inhibition concentration of thedifferentiation inhibitor.

In the methods and compositions, the stemness driver can be a gsk3-betainhibitor, a gsk3-beta inhibitor derivative, a wnt agonist, a wntagonist derivative, or a pharmaceutically acceptable salt of any of theforegoing. In the method and composition, the differentiation inhibitorcan be a notch agonist; a notch agonist derivative; an hdac inhibitor;an hdac inhibitor derivative, or a pharmaceutically acceptable salt ofany of the foregoing. In methods and compositions, the stemness drivercan be selected from the group consisting of CHIR99021, LY2090314,lithium, A1070722, BML-284 and SKL2001.

In the methods and compositions, the differentiation inhibitor can be aNotch agonist or an HDAC inhibitor selected from the group consisting ofvalproic acid, SAHA and Tubastatin A

Also dscribed herein is a method of treating a subject who has, or is atrisk of developing, hearing loss. The method can includetrans-tympanically administering to a cochlear tissue of the subject acomposition comprising at least one stem cell proliferator. The at leastone stem cell proliferator can include at least one of a stemness driverand a differentiation inhibitor. The at least one stem cell proliferatorcan include both a stemness driver and a differentiation inhibitor.

The stemness driver can be CHIR99021. The differentiation inhibitor canbe valproic acid. In some methods, the cochlear tissue can be furthercontacted with epidermal growth factor, basic fibroblast growth factor,insulin-like growth factor 1, pVc, and 616452.

Also described herein is a method of generating Myo7a+ cochlear cells.The method can include contacting Lgr5+ cochlear cells with acomposition comprising a stemness driver and a differentiationinhibitor, thereby generating an expanded population of Lgr5+ cells; andcontacting the expanded population of Lgr5+ cells with a notch inhibitorand a stemness driver, thereby generating Myo7a+ cochlear cells. Thestemness driver can be CHIR99021. The Differentiation Inhibitor canvalproic acid. The notch inhibitor can be DAPT.

In some aspects, the invention includes compounds comprising one or moreof the compounds described herein, such as a stem cell proliferator. Insome aspects, the invention encompasses a kit, e.g., a kit comprising akinase inhibitor. In some aspects, the kit includes instructions.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIGS. 1A-B: Expansion of Lgr5-GFP inner ear supporting cells in multipleconditions. FIG. 1A: Brightfield and GFP fluorescence images of Lgr5-GFPinner ear progenitor cells cultured for 10 days in media containing EGF,bFGF and IGF-1. FIG. 1B: Lgr5-GFP cells were cultured in conditions withthe addition of CHIR and VPA. Scale bars 100 μm.

FIGS. 2A-C: Small molecules (CHIR99021 and VPA) promote the expansion ofinner ear progenitor cells. FIG. 2A: FACS histogram images of Lgr5-GFPcells under multiple conditions. FIG. 2B: Quantification of cellproliferation and GFP expression of cells as shown in FIG. 2A. FIG. 2C:Corresponding quantification of cells.

FIG. 3: Addition of pVc increases cell proliferation of Lgr5 inner earprogenitor cells. Scale bars: 400 μm.

FIG. 4: Increasing bFGF concentration promotes the proliferation of Lgr5inner ear progenitor cells. Scale bars: 400 μm.

FIGS. 5A-B: Further screening of supportive factors for Lgr5 inner earprogenitor cells. TTNPB increased cell proliferation but didn't increaseGFP expression. In the presence of pVc, CHIR is critical for cellproliferation and GFP expression, EGF, bFGF is important for cellproliferation but less important for GFP expression. VPA is importantfor GFP expression. IGF shows a marginal beneficial effect in promotingcell proliferation and GFP expression. FIG. 5A: Cell number. FIG. 5B:GFP+ percentage.

FIG. 6: Further screening of supportive factors for Lgr5 inner earprogenitor cells. Additional screening with major signaling pathwaymodulators demonstrates the manipulation of these singling pathways doesnot promote the expression of Lgr5-GFP. Small molecules used inscreening include: PD0325901 (MEK inhibitor), VX745 (p38 inhibitor),JNK-IN8 (JNK inhibitor), Tofacitinib (JAK inhibitor), PH797804 (p38inhibitor), Rapamycin (mTor inhibitor), LY294002 (PI3K inhibitor), SC79(AKT activator), PKC412 (PKC inhibitor), FR180209 (Insulin Receptorinhibitor), LDN193189 (BMP inhibitor), BMS536924 (Insulin andinsulin-like growth factor-1 receptor inhibitor), 5IT (Increase betacell proliferation) and 616452 (Tgfb, ALK5 inhibitor).

FIG. 7: The addition of 616452 on EFICVP increases the intensity ofLgr5-GFP and the size of colonies. Scale bars: 400 μm.

FIGS. 8A-C depict characterization of culture conditions to promoteproliferation of inner ear progenitor cells. FIG. 8A shows GFPfluorescence and bright-field images of single inner ear epithelialcells for 10 days in the presence of EGF, bFGF, IGF1 (EFI); EFI andCHIR99021, VPA, pVc, 616452 (EFICVP6). FIG. 8B shows quantification ofLgr5-GFP expression, cell proliferation (number of live cells), andnumber of GFP⁺ cells in inner ear epithelial cells cultured for 10 daysin multiple conditions. Cell colonies were dissociated into single cellsusing trypsin. Total number of cells were counted using a hemocytometer.The cells were then stained with propidium iodide (PI) and analyzedusing a flow cytometer for Lgr5-GFP expression. Number of GFP⁺ cellswere calculated by multiplying the total number of cells with percentageof GFP⁺ cells. EFICVP6 indicates culture conditions containing all thefactors including EGF, bFGF, IGF1, CHIR, VPA, pVc, and 616452, whichshowed the best results in supporting cell proliferation and GFPexpression. Each individual factor was then removed from the culturemedia and tested. Removing bFGF or CHIR from the media greatly impactedcell proliferation and removing CHIR also greatly impacted GFPexpression. Removing EGF, 616452 showed greater impact on cellproliferation, while removing VPA or pVc showed greater impact on GFPmaintenance. Removing IGF-1 showed marginal effect on cell proliferationor GFP maintenance. These results suggest bFGF and CHIR are important,and other factors are also important, in promoting cell proliferationand GFP expression. FIG. 8C shows GFP fluorescence and brightfieldimages of cultures as shown in (b). Scale bars: 200 m (a) and 400 μm(c).

FIGS. 9A-F demonstrate how small molecules promote the maintenance ofinner ear progenitor cells. FIG. 9A shows GFP expression of culturedinner ear epithelial cells in multiple conditions. W: Wnt3a. R:R-Spondin 1. FIG. 9B_shows histogram images of GFP expression of cellscultured in multiple conditions. FIG. 9C shows GFP fluorescence andbrightfield images of Lgr5-GFP inner ear epithelial cells culture for 7days in conditions as indicated. FIG. 9D shows GFP fluorescence andbrightfield images of Atoh1-GFP inner ear epithelial cells 7 days inculture conditions as indicated. GFP+ cells were differentiated cells.Results show VPA inhibited the differentiation of inner ear progenitorcells towards Atoh1 positive hair cells. FIG. 9E shows a screen forsupportive factors for inner ear progenitor cells. Shown are GFPexpression of Lgr5-GFP inner ear progenitor cells cultured in multipleconditions. Small molecules were added based on control (EFICV)condition. Laminin 511 was added into Matrigel. Two batches of cellswere used for screening as shown as Exp 1 and Exp 2. Results show pVcpromoted GFP expression from Lgr5-GFP inner ear progenitor cells. FIG.9F shows fluorescence and brightfield images of Lgr5-GFP cells culturedin conditions as indicated. Shown are cells at day 10 of passage 2.616452 enables the passage of cultured inner ear progenitor cells. Scalebars: 400 μm.

FIG. 10 demonstrates the expansion of single sorted Lgr5-GFP cells.Left, sorted GFP-High cells grow into large colonies uniformly expresshigh level of GFP. Right, GFP-Low cells grow into colonies containingGFP-high (large arrow), GFP-low (arrow head) and GFP negative (smallarrow) colonies.

FIG. 11 depicts differentiation of expanded inner ear progenitors inmultiple conditions. qPCR was performed to measure Myo7a expressionfollowing 6 days of differentiation. The condition without growthfactors (EGF, bFGF, IGF) or small molecules (616452, pVC and VPA) giveshighest Myo7a expression.

FIGS. 12A-B demonstrate that cultured inner ear progenitor cellsgenerate hair cells in vitro. FIG. 12A shows that the combination withGsk3β inhibitor (e.g. CHIR) and Notch inhibitor (e.g. DAPT) induce thegeneration of Myo7a positive and Prestin positive outer hair cells(upper panel) and Myo7a positive and Prestin negative inner hair cells(lower panel). FIG. 12B shows that in the presence of Wnt inhibitorIWP-2, hair cell generation is rarely observed from cultured inner earprogenitor cells. Scale bars: 100 μm.

FIGS. 13A-C depict In vitro culture of Lgr5-GFP inner ear progenitorcells from adult mouse. FIG. 13A: Isolated cochlear from 9-week old micewas cultured in the presence of EFICVP6 for 9 days. Showing out-growthof Lgr5-GFP cells. FIG. 13B: The same culture at day 13. Showingexpansion of Lgr5-GFP cells. FIG. 13C: Passage 2 of the same culture atday 5 after passage (Day 19). All scale bars: 100 μm. Specifically,cochlear was isolated from 9-week old adult mice, directly mixed withMatrigel and plated at the center of well of a 24-well plate. Cellculture medium in the presence of EGF, bFGF, IGF-1, CHIR99021, VPA,L-Ascorbic acid 2-phosphate (pVc) and a TGF-β RI Kinase Inhibitor II(616452) was added following gelation of Matrigel. The tissue wascultured for 2 weeks until Lgr5-GFP cells expanded and form out-growth.After 2 weeks (Day 14) the tissue was then dissociated using trypsin,re-plated and further cultured. Results comparing Day 9 to Day 13 showthat our culture system can be used to support the growth and expansionof adult Lgr5-GFP cells from inner ear epithelium. The results also showthat using these culture conditions Lgr5⁺ cells from adult cochlea canbe passaged (FIG. 13C).

FIG. 14. Expansion of Lgr5-GFP inner ear cells from adult mice. InEFICVP6 condition, cell grow slowly. The addition of TTNPB increase cellproliferation and formation of GFP⁺ colonies.

FIG. 15. Expansion of Lgr5-GFP+ inner ear cells from adult mice. Cellswere cultured in conditions containing EGF, bFGF, IGF1, CHIR, VPA, pVc,616452 and TTNPB. Images were taken at the same field on day 2 and day 5in culture. Brightfield and GFP fluorescence images were shown.

FIG. 16A: Percentage cumulative release of CHIR from poloxamer 407 basedhydrogel formulation in a dialysis bag set up. FIG. 16B: Percentagecumulative release of VPA from poloxamer 407 based hydrogel formulationin a dialysis bag set up.

FIG. 17A: Cumulative release of CHIR from poloxamer 407 based hydrogelformulation in a dialysis bag set up. Initial loadings of CHIR and VPAwere 41.7 μg and 2.63 mg per 30 ul, respectively. FIG. 17B: Cumulativerelease of VPA from poloxamer 407 based hydrogel formulation in adialysis bag set up. Initial loadings of CHIR and VPA were 41.7 μg and2.63 mg per 30 ul, respectively.

FIG. 18: Hearing recovery in CBA/CaJ mice. Animals treated with VPA/CHIR(n=7) showed significant recovery across all tested frequencies. Animalstreated with LY411575 (n=8) showed significant recovery at 10 kHz, 28.3KHz, and 40 kHz. Recovery did not occur in animals treated with controlvehicle injections (n=8). [*=p<0.05]

Corresponding reference characters indicate corresponding partsthroughout the drawings.

Definitions

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration” refers to introducing a substance into a subject. Insome embodiments, administration is auricular, intraauricular,intracochlear, Intravestibular, or transtympanically, e.g., byinjection. In some embodiments, administration is directly to the innerear, e.g. injection through the round window, otic capsule, orvestibular canals. In some embodiments, administration is directly intothe inner ear via a cochlear implant delivery system. In someembodiments, the substance is injected transtympanically to the middleear. In certain embodiments “causing to be administered” refers toadministration of a second component after a first component has alreadybeen administered (e.g., at a different time and/or by a differentactor).

An “antibody” refers to an immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in theexpression or activity of a target gene, protein, or a pathway,respectively. Therefore, an agonist can bind to and activate its cognatereceptor in some fashion, which directly or indirectly brings about thisphysiological effect on the target gene or protein. An agonist can alsoincrease the activity of a pathway through modulating the activity ofpathway components, for example, through inhibiting the activity ofnegative regulators of a pathway. Therefore, a “Wnt agonist” can bedefined as an agent that increases the activity of Wnt pathway, whichcan be measured by increased TCF/LEF-mediated transcription in a cell.Therefore, a “Wnt agonist” can be a true Wnt agonist that bind andactivate a Frizzled receptor family member, including any and all of theWnt family proteins, an inhibitor of intracellular beta-catenindegradation, and activators of TCF/LEF. A “Notch agonist” can be definedas an agent that increase the activity of Notch pathway, which can bedetermined by measuring the transcriptional activity of Notch.

An “antagonist” refers to an agent that binds to a receptor, and whichin turn decreases or eliminates binding by other molecules.

“Anti-sense” refers to a nucleic acid sequence, regardless of length,that is complementary to the coding strand or mRNA of a nucleic acidsequence. Antisense RNA can be introduced to an individual cell, tissueor organanoid. An anti-sense nucleic acid can contain a modifiedbackbone, for example, phosphorothioate, phosphorodithioate, or othermodified backbones known in the art, or may contain non-naturalinternucleoside linkages.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence capable of hybridizing with another nucleic acidsequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

“Auricular administration” refers to a method of using a catheter orwick device to administer a composition across the tympanic membrane tothe inner ear of the subject. To facilitate insertion of the wick orcatheter, the tympanic membrane may be pierced using a suitably sizedsyringe or pipette. The devices could also be inserted using any othermethods known to those of skill in the art, e.g., surgical implantationof the device. In particular embodiments, the wick or catheter devicemay be a stand-alone device, meaning that it is inserted into the ear ofthe subject and then the composition is controllably released to theinner ear. In other particular embodiments, the wick or catheter devicemay be attached or coupled to a pump or other device that allows for theadministration of additional compositions. The pump may be automaticallyprogrammed to deliver dosage units or may be controlled by the subjector medical professional.

“Biocompatible Matrix” as used herein is a polymeric carrier that isacceptable for administration to humans for the release of therapeuticagents. A Biocompatible Matrix may be a biocompatible gel or foam.

“Cell Aggregate” as used herein shall mean a body cells in the Organ ofCorti that have proliferated to form a cluster of a given cell type thatis greater than 40 microns in diameter and/or produced a morphology inwhich greater than 3 cell layers reside perpendicular to the basilarmembrane. A “Cell Aggregate” can also refer a process in which celldivision creates a body of cells that cause one or more cell types tobreach the reticular lamina, or the boundary between endolymph andperilymph

“Cell Density” as used herein in connection with a specific cell type isthe mean number of that cell type per area in a RepresentativeMicroscopy Sample. The cell types may include but are not limited toLgr5⁺ cells, hair cells, or supporting cells. The Cell Density may beassessed with a given cell type in a given organ or tissue, includingbut not limited to the cochlea or Organ of Corti. For instance, theLgr5⁺ Cell Density in the Organ of Corti is the Cell Density of Lgr5⁺cells as measured across the Organ of Corti. Typically, supporting cellsand Lgr5⁺ cells will be enumerated by taking cross sections of the Organof Corti. Typically, hair cells will be enumerated by looking down atthe surface of the Organ of Corti, though cross sections may be used insome instances, as described in a Representative Microscopy Sample.Typically, Cell Density of Lgr5⁺ cells will be measured by analyzingwhole mount preparations of the Organ of Corti and counting the numberof Lgr5 cells across a given distance along the surface of theepithelia, as described in a Representative Microscopy Sample. Haircells may be identified by their morphological features such as bundlesor hair cell specific stains (e.g., Myosin VIIa, Prestin, vGlut3,Pou4f3, Espin, conjugated-Phalloidin, PMCA2, Ribeye, Atoh1, etc). Lgr5+cells may be identified by specific stains or antibodies (e.g. Lgr5-GFPtransgenic reporter, anti-Lgr5 antibody, etc.)

“CHIR99021” is a chemical composition having the chemical formulaC₂₂H₁₈Cl₂N₈ and the following alternate names: CT 99021;6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile.Its chemical structure is as follows:

“Cochlear Concentration” as used herein will be the concentration of agiven agent as measured through sampling cochlear fluid. Unlessotherwise noted, the sample should contain a substantial enough portionof the cochlear fluid so that it is approximately representative of theaverage concentration of the agent in the cochlea. For example, samplesmay be drawn from a vestibular canal, and a series of fluid samplesdrawn in series such that individual samples are comprised of cochlearfluid in specified portions of the cochlea

“Complementary nucleic acid sequence” refers to a nucleic acid sequencecapable of hybridizing with another nucleic acid sequence comprised ofcomplementary nucleotide base pairs.

“Cross-Sectional Cell Density” as used herein in connection with aspecific cell type is the mean number of that cell type per area ofcross section through a tissue in a Representative Microscopy Sample.Cross sections of the Organ of Corti can also be used to determine thenumber of cells in a given plane. Typically, hair cells Cross-sectionalCell Density will be measured by analyzing whole mount preparations ofthe Organ of Corti and counting the number of hair cells across a givendistance in cross sections taken along a portion of the epithelia, asdescribed in a Representative Microscopy Sample. Typically,Cross-sectional Cell Density of Lgr5⁺ cells will be measured byanalyzing whole mount preparations of the Organ of Corti and countingthe number of Lgr5⁺ cells across a given distance in cross sectionstaken along a portion of the epithelia, as described in a RepresentativeMicroscopy Sample. Hair cells may be identified by their morphologicalfeatures such as bundles or hair cell specific stains (suitable stainsinclude e.g., Myosin VIIa, Prestin, vGlut3, Pou4f3,conjugated-Phalloidin, PMCA2, Atoh1, etc.). Lgr5⁺ cells may beidentified by specific stains or antibodies (suitable stains andantibodies include fluorescence in situ hybridization of Lgr5 mRNA,Lgr5-GFP transgenic reporter system, anti-Lgr5 antibodies, etc.).

“Decreasing” refers to decreasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99 or 100%, for example, as compared to the level of reference.

“Decreases” also means decreases by at least 1-fold, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000-fold or more, for example, as compared to the level of areference.

“Differentiation Inhibitor” as used herein is an agent which may inhibitdifferentiation of an inner ear stem cell into an inner ear hair cell.Some differentiation inhibitors maintain expression of post-natal StemCell Markers. Some Differentiation Inhibitors include, withoutlimitation, Notch agonists and HDAC inhibitors.

“Differentiation Period” as used herein is the duration of time in whichthere is an Effective Stemness Driver Concentration without an EffectiveDifferentiation Inhibition Concentration.

“Effective Concentration” may be the Effective Stemness DriverConcentration for a Stemness Driver or the Effective DiffusionInhibition Concentration for a Diffusion Inhibitor.

“Effective Differentiation Inhibition Concentration” is the minimumconcentration of a Differentiation Inhibitor that does not allow morethan a 50% increase in the fraction of the total population of cellsthat are hair cells at the end of the Stem Cell Proliferation Assaycompared to the start of the Stem Cell Proliferation Assay In measuringthe Effective Differentiation Inhibition Concentration, a Hair Cellstain for cells may be used with flow cytometry to quantify hair cellsfor a mouse strain that is not an Atoh1-GFP mouse. Alternatively, andAtoh1-GFP mouse strain may be used.

“Effective Release Rate” (mass/time) as used herein is the EffectiveConcentration (mass/volume)*30 uL/1 hour.

“Effective Stemness Driver Concentration” is the minimum concentrationof a Stemness Driver that induces at least 1.5-fold increase in numberof LGR5+ cells in a Stem Cell Proliferation Assay compared to the numberof Lgr5+ cells in a Stem Cell Proliferation Assay performed without theStemness Driver and with all other components present at the sameconcentrations.

“Eliminate” means to decrease to a level that is undetectable.

“Engraft” or “engraftment” refers to the process of stem or progenitorcell incorporation into a tissue of interest in vivo through contactwith existing cells of the tissue. “Epithelial progenitor cell” refersto a multipotent cell which has the potential to become restricted tocell lineages resulting in epithelial cells.

“Epithelial stem cell” refers to a multipotent cell which has thepotential to become committed to multiple cell lineages, including celllineages resulting in epithelial cells.

“Fragment” refers to a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

“GSK3beta,” “GSK3β,” and “GSK3B” as used interchangeably herein areacronyms for glycogen synthase kinase 3 beta,

“GSK3beta inhibitor” is a composition that inhibits the activity ofGSK3beta.

“HDAC” as used herein is an acronym for histone deacetylase.

“HDAC inhibitor” is a composition that inhibits the activity of HDAC.

“Hybridize” refers to pairing to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

An “inhibitor” refers to an agent that causes a decrease in theexpression or activity of a target gene or protein, respectively. An“antagonist” can be an inhibitor, but is more specifically an agent thatbinds to a receptor, and which in turn decreases or eliminates bindingby other molecules.

As used herein, an “inhibitory nucleic acid” is a double-stranded RNA,RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portionthereof, or a mimetic thereof, that when administered to a mammaliancell results in a decrease in the expression of a target gene.Typically, a nucleic acid inhibitor comprises at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. Typically, expression of a target gene is reduced by 10%, 25%,50%, 75%, or even 90-100%.

“In Vitro Lgr5 activity” refers to the level of expression or activityof Lgr5 in an in vitro population of cells. It may be measured, forexample, in cells derived from a Lgr5-GFP expressing mouse such as aB6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (also known asLgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No: 008875)by dissociating cells to single cells, staining with propidium iodide(PI), and analyzing the cells using a flow cytometer for Lgr5-GFPexpression. Inner ear epithelial cells from wild-type (non-Lgr5-GFP)mice that passing the same culturing and analyzing procedures can beused as a negative control. Typically, two population of cells are shownin the bivariate plot with GFP/FITC as one variable, which include bothGFP positive and GFP negative populations. Lgr5-positive cells areidentified by gating GFP positive cell population. The percentage ofLgr5-positive cells are measured by gating GFP positive cell populationagainst both GFP negative population and the negative control. Thenumber of Lgr5-positive cells is calculated by multiplying the totalnumber of cells by the percentage of Lgr5-positive cells. For cellsderived from non-Lgr5-GFP mice, Lgr5 activity can be measured using ananti-Lgr5 antibody or quantitative-PCR on the Lgr5 gene.

“In Vivo Lgr5 activity” as used herein is the level of expression oractivity of Lgr5 in a subject. It may be measured, for example, byremoving an animal's inner ear and measuring Lgr5 protein or Lgr5 mRNA.Lgr5 protein production can be measured using an anti-Lgr5 antibody tomeasure fluorescence intensity as determined by imaging cochlearsamples, where fluorescence intensity is used as a measure of Lgr5presence. Western blots can be used with an anti-Lgr5 antibody, wherecells can be harvested from the treated organ to determine increases inLgr5 protein. Quantitative-PCR or RNA in situ hybridization can be usedto measure relative changes in Lgr5 mRNA production, where cells can beharvested from the inner ear to determine changes in Lgr5 mRNA.Alternatively, Lgr5 expression can be measured using an Lgr5 promoterdriven GFP reporter transgenic system, where the presence or intensityGFP fluoresce can be directly detected using flow cytometry, imaging, orindirectly using an anti-GFP antibody.

“Increases” also means increases by at least 1-fold, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000-fold or more, for example, as compared to the level of a ascompared to the level of a reference standard.

“Increasing” refers to increasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99, 100% or more, for example, as compared to the level of areference.

“Intraauricular administration” refers to administration of acomposition to the middle or innerear of a subject by directly injectingthe composition.

“Intracochlear” administration refers to direct injection of acomposition across the tympanic membrane and across the round windowmembrane into the cochlea.

“Intravestibular” administration refers to direct injection of acomposition across the tympanic membrane and across the round windowmembrane into the vestibular organs.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings.

“Lgr5” is an acronym for the Leucine-rich repeat-containing G-proteincoupled receptor 5, also known as G-protein coupled receptor 49 (GPR49)or G-protein coupled receptor 67 (GPR67). It is a protein that in humansis encoded by the Lgr5 gene.

“Lgr5 activity” is defined as the level of activity of Lgr5 in apopulation of cells. In an in vitro cell population, Lgr5 activity maybe measured in an in vitro Lgr5 Activity assay. In an in vivo cellpopulation, Lgr5 activity may be measured in an in vivo Lgr5 Activityassay.

“Lgr5⁺ cell” or “Lgr5-positive cell” as used herein is a cell thatexpresses Lgr5. “Lgr5⁻ cell” as used herein is a cell that is not Lgr5⁺.

“Lineage Tracing” as used herein is using a mouse line that enables fatetracing of any cell that expresses a target gene at the time of reporterinduction. This can include hair cell or supporting cells genes (Sox2,Lgr5, MyosinVIIa, Pou4f3, etc). For example, lineage tracing may use anLgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, which uponinduction, allows one to trace the fate of cells that expressed Lgr5 atthe time of induction. By further example, Lgr5 cells can be isolatedinto single cells and cultured in a Stem Cell Proliferation Assay togenerate colonies, then subsequently differentiated in a DifferentiationAssay and analyzed for cell fate by staining for hair cell and/orsupporting cell proteins and determining the reporter colocalizationwith either hair cell or supporting cell staining to determine the Lgr5cells' fate. In addition, lineage tracing can be performed in cochlearexplants to track supporting cell or hair cell fate within the intactorgan after treatment. For example, Lgr5 cell fate can be determined byisolating the cochlea from a Lgr5-EGFP-IRES-creERT2 mouse crossed with areporter mouse, and inducing the reporter in Lgr5 cells before or duringtreatment. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determining the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. In addition, lineage tracing can beperformed in vivo track supporting cell or hair cell fate within theintact organ after treatment. For example, Lgr5 cell fate can bedetermined inducing a reporter in an Lgr5-EGFP-IRES-creERT2 mousecrossed with a reporter mouse, treating the animal, then isolating thecochlea. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determinning the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. Lineage tracing may be performed usingalternative reporters of interest as is standard in the art.

“Mammal” refers to any mammal including but not limited to human, mouse,rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

“Mean Release Time” as used herein is the time in which one-half of anagent is released into phosphate buffered saline from a carrier in aRelease Assay.

“Native Morphology” as used herein is means that tissue organizationlargely reflects the organization in a healthy tissue. For example,“Native Morphology” for a cochlea means that hair cells are surroundedby supporting cells in a rosette pattern formed by lateral inhibition ofNotch, hair cells do not contact each other, 2-3 cell layers form theorgan of Corti epithelia, and cells do not breach the reticular lamina(ie. do not breach the border between endolymph and perilymph).

“Non-human mammal”, as used herein, refers to any mammal that is not ahuman.

“Notch activity assay” as used herein refers to an assay to determinethe Notch activity in a population using standard techniques, such asdetermining the expression of Hes5/Hes1 using, for example, qPCR.

As used in relevant context herein, the term “number” of cells can be 0,1, or more cells.

“Organ of Corti” as used herein refers to the sensory cells (inner andouter hair cells) of the hearing organ located in the cochlea.

“Organoid” or “epithelial organoid” refers to a cell cluster oraggregate that resembles an organ, or part of an organ, and possessescell types relevant to that particular organ.

“Population” of cells refers to any number of cells greater than 1, butis preferably at least 1×10³ cells, at least 1×10⁴ cells, at least atleast 1×10⁵ cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least1×10⁸ cells, at least 1×10⁹ cells, or at least 1×10¹⁰ cells.

“Progenitor cell” as used herein refers to a cell that, like a stemcell, has the tendency to differentiate into a specific type of cell,but is already more specific than a stem cell and is pushed todifferentiate into its “target” cell.

“Proliferation Period” as used herein is the duration of time in whichthere is an Effective Sternness Driver Concentration and aDifferentiation Inhibition Concentration of a Differentiation Inhibitor.

“Reference” means a standard or control condition (e.g., untreated witha test agent or combination of test agents).

“Release Assay” as used herein is a test in which the rate of release ofan agent from a Biocompatible Matrix through dialysis membrane to asaline environment. An exemplary Release Assay may be performed byplacing 30 microliters of a composition in 1 ml Phosphate BufferedSaline inside saline dialysis bag with a suitable cutoff, and placingthe dialysis bag within 10 mL of Phosphate Buffered Saline at 37 C. Thedialysis membrane size may be chosen based on agent size in order toallow the agent being assessed to exit the membrane. For small moleculerelease, a 3.5-5 kDa cutoff may be used. The agent may be a StemnessDriver, Differentiation Inhibitor, or other agent. The Release Rate fora composition may change over time and may be measured in 1 hourincrements.

“Representative Microscopy Sample” as used herein describes a sufficientnumber of fields of view within a cell culture system, a portion ofextracted tissue, or an entire extracted organ that the average featuresize or number being measured can reasonably be said to represent theaverage feature size or number if all relevant fields were measured. Forexample, in order to assess the hair cell counts at a frequency range onthe Organ of Corti, ImageJ software (NIH) can used to measure the totallength of cochlear whole mounts and the length of individual countedsegments. The total number of inner hair cells, outer hair cells, andsupporting cells can be counted in the entire or fraction of any of thefour cochlear segments of 1200-1400 m (apical, mid-apical, mid-basal,and basal) at least 3 fields of view at 100 μm field size would bereasonably considered a Representative Microscopy Sample. ARepresentative Microscopy sample can include measurements within a fieldof view, which can be measured as cells per a given distance. ARepresentative Microscopy sample can be used to assess morphology, suchas cell-cell contacts, cochlear architecture, and cellular components(e.g., bundles, synapses).

“Rosette Patterning” is a characteristic cell arrangement in the cochleain which <5% hair cells are adjacent to other hair cells.

The term “sample” refers to a volume or mass obtained, provided, and/orsubjected to analysis. In some embodiments, a sample is or comprises atissue sample, cell sample, a fluid sample, and the like. In someembodiments, a sample is taken from (or is) a subject (e.g., a human oranimal subject). In some embodiments, a tissue sample is or comprisesbrain, hair (including roots), buccal swabs, blood, saliva, semen,muscle, or from any internal organs, or cancer, precancerous, or tumorcells associated with any one of these. A fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. A body tissue can include, but is not limited to, brain, skin,muscle, endometrial, uterine, and cervical tissue or cancer,precancerous, or tumor cells associated with any one of these. In anembodiment, a body tissue is brain tissue or a brain tumor or cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a “sample” is a “primary sample” in that it is obtainedfrom a source (e.g., a subject); in some embodiments, a “sample” is theresult of processing of a primary sample, for example to remove certainpotentially contaminating components and/or to isolate or purify certaincomponents of interest.

“Self-renewal” refers to the process by which a stem cell divides togenerate one (asymmetric division) or two (symmetric division) daughtercells with development potentials that are indistinguishable from thoseof the mother cell. Self-renewal involves both proliferation and themaintenance of an undifferentiated state.

“siRNA” refers to a double stranded RNA. Optimally, an siRNA is 18, 19,20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang atits 3′ end. These dsRNAs can be introduced to an individual cell orculture system. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

“Stem cell” refers to a multipotent cell having the capacity toself-renew and to differentiate into multiple cell lineages.

“Stem Cell Differentiation Assay” as used herein is an assay todetermine the differentiation capacity of stem cells. In an exemplaryStem Cell Differentiation Assay, the number of cells for an initial cellpopulation is harvested from a Atoh1-GFP mouse between the age of 3 to 7days, by isolating the Organ of Corti sensory epithelium, dissociatingthe epithelium into single cells, and passing the cells through a 40 umcell strainer. Approximately 5000 cells are entrapped in 40 μl ofculture substrate (for example: Matrigel (Corning, Growth FactorReduced)) and placed at the center of wells in a 24-well plate with 500μl of an appropriate culture media, growth factors and agent beingtested. Appropriate culture media and growth factors include AdvancedDMEM/F12 with media Supplements (1×N2, 1×B27, 2 mM Glutamax, 10 mMHEPES, 1 mM N-acetylcysteine, and 100 U/ml Penicillin/100 μg/mlStreptomycin) and growth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50ng/ml IGF-1) as well as the agent(s) being assessed are added into eachwell. Cells are cultured for 10 days in a standard cell cultureincubator at 37° C. and 5% CO₂, with media change every 2 days. Thesecells are then cultured by removing the Stem Cell Proliferation Assayagents and replacing with Basal culture media and molecules to drivedifferentiation. An appropriate Basal culture media is Advanced DMEM/F12supplemented with 1×N2, 1×B27.2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin andappropriate molecules to drive differentiation are 3 μM CHIR99021 and 5μM DAPT for 10 days, with media change every 2 days. The number of haircells in a population may be measured by using flow cytometry for GFP.Hair cell differentiation level can further be assessed using qPCR tomeasure hair cell marker (e.g., Myo7a) expression level normalized usingsuitable and unregulated references or housekeeping genes (e.g., Hprt).Hair cell differentiation level can also be assessed by immunostainingfor hair cell markers (eg. Myosin7a, vGlut3, Espin, PMCAs, Ribeye,conjugated-phalloidin, Atoh1, Pou4f3, etc). Hair cell differentiatonlevel can also be assessed by Western Blot for Myosin7a, vGlut3, Espin,PMCAs, Prestin, Ribeye, Atoh1, Pou4f3.

“Stem Cell Assay” as used herein is an assay in which a cell or a cellpopulation are tested for a series of criteria to determine whether thecell or cell population are stem cells or enriched in stem cells or stemcell markers. In a stem cell assay, the cell/cell population are testedfor stem cell characteristics such as expression of Stem Cell Markers,and further optionally are tested for stem cell function, including thecapacity of self-renewal and differentiation.

“Stem Cell Proliferator” as used herein is a composition that induces anincrease in a population of cells which have the capacity forself-renewal and differentiation.

“Stem Cell Proliferation Assay” as used herein is an assay to determinethe capacity for agent(s) to induce the creation of stem cells from astarting cell population. In an exemplary Stem Cell Proliferation Assay,the number of cells for an initial cell population is harvested from aLgr5-GFP mouse such as a B6.129P2-Lgr5tm1(cre/ERT2)Cle/J mouse (alsoknown as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No:008875) between the age of 3 to 7 days, by isolating the Organ of Cortisensory epithelium and dissociating the epithelium into single cells.Approximately 5000 cells are entrapped in 40 μl of culture substrate(for example: Matrigel (Corning, Growth Factor Reduced)) and placed atthe center of wells in a 24-well plate with 500 μl of an appropriateculture media, growth factors and agent being tested. Appropriateculture media and growth factors include Advanced DMEM/F12 with mediaSupplements (1×N2, 1×B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin) andgrowth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50 ng/ml IGF-1) as wellas the agent(s) being assessed are added into each well. Cells arecultured for 10 days in a standard cell culture incubator at 37° C. and5% CO₂, with media change every 2 days. The number of Lgr5⁺ cells isquantified by counting the number of cells identified as Lgr5+ in an InVitro Lgr5 activity assay. The fraction of cells that are Lgr5⁺ isquantified by dividing the number of cells identified as Lgr5⁺ in a cellpopulation by the total number of cells present in the cell population.The average Lgr5⁺ activity of a population is quantified by measuringthe average mRNA expression level of Lgr5 of the population normalizedusing suitable and unregulated references or housekeeping genes (e.g.,Hprt). The number of hair cells in a population may be measured bystaining with hair cell marker (e.g., MyosinVIIa), or using anendogenous reporter of hair cell genes (eg. Pou4f3-GFP, Atoh1-nGFP) andanalyzing using flow cytometry. The fraction of cells that are haircells is quantified by dividing the number of cells identified as haircells in a cell population by the total number of cells present in thecell population. Lgr5 activity can be measured by qPCR.

“Stem Cell Markers” as used herein can be defined as gene products (e.g.protein, RNA, etc) that specifically expressed in stem cells. One typeof stem cell marker is gene products that are directly and specificallysupport the maintenance of stem cell identity. Examples include Lgr5 andSox2. Additional stem cell markers can be identified using assays thatwere described in the literatures. To determine whether a gene isrequired for maintenance of stem cell identity, gain-of-function andloss-of-function studies can be used. In gain-of-function studies, overexpression of specific gene product (the stem cell marker) would helpmaintain the stem cell identity. While in loss-of-function studies,removal of the stem cell marker would cause loss of the stem cellidentity or induced the differentiation of stem cells. Another type ofstem cell marker is gene that only expressed in stem cells but does notnecessary to have specific function to maintain the identity of stemcells. This type of markers can be identified by comparing the geneexpression signature of sorted stem cells and non-stem cells by assayssuch as micro-array and qPCR. This type of stem cell marker can be foundin the literature. (e.g. Liu Q. et al., Int J Biochem Cell Biol. 2015March; 60:99-111. http://www.ncbi.nlm.nih.gov/pubmed/25582750).Potential stem cell markers include Ccdc121, Gdf10, Opcm1, Phex, etc.The expression of stem cell markers such as Lgr5⁺ or Sox2 in a givencell or cell population can be measure using assays such as qPCR,immunohistochemistry, western blot, and RNA hybridization. Theexpression of stem cell markers can also be measured using transgeniccells express reporters which can indicate the expression of the givenstem cell markers, e.g. Lgr5-GFP or Sox2-GFP. Flow cytometry analysiscan then be used to measure the activity of reporter expression.Fluorescence microscopy can also be used to directly visualize theexpression of reporters. The expression of stem cell markers may furtherbe determined using microarray analysis for global gene expressionprofile analysis. The gene expression profile of a given cell populationor purified cell population can be compared with the gene expressionprofile of the stem cell to determine similarity between the 2 cellpopulations. Stem cell function can be measured by colony forming assayor sphere forming assay, self-renewal assay and differentiation assay.In colony (or sphere) forming assay, when cultured in appropriateculture media, the stem cell should be able to form colonies, on cellculture surface (e.g. cell culture dish) or embedded in cell culturesubstrate (e.g. Matrigel) or be able to form spheres when cultured insuspension. In colony/sphere forming assay, single stem cells are seededat low cell density in appropriate culture media and allowed toproliferate for a given period of time (7-10 days). Colony formed arethen counted and scored for stem cell marker expression as an indicatorof stemness of the original cell. Optionally, the colonies that formedare then picked and passaged to test its self-renewal anddifferentiation potential. In self-renewal assay, when cultured inappropriate culture media, the cells should maintain stem cell marker(e.g. Lgr5) expression over at least one (e.g. 1, 2, 3, 4, 5, 10, 20,etc) cell divisions. In a Stem Cell Differentiation Assay, when culturedin appropriate differentiation media, the cells should be able togenerate hair cell which can be identified by hair cell markerexpression measured by qPCR, immunostaining, western blot, RNAhybridization or flow cytometry.

“Stemness Driver” as used herein is a composition that inducesproliferation of LGR5⁺ cells, upregulates Lgr5 in cells, or maintainsLgr5 expression in cells, while maintaining the potential forself-renewal and the potential to differentiate into hair cells.Generally, sternness drivers upregulate at least one biomarker ofpost-natal stem cells. Sternness Drivers include but are not limited toWnt agonists and GSK3Beta inhibitors.

“Subject” includes humans and mammals (e.g., mice, rats, pigs, cats,dogs, and horses). In many embodiments, subjects are be mammals,particularly primates, especially humans. In some embodiments, subjectsare livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. In someembodiments (e.g., particularly in research contexts) subject mammalswill be, for example, rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like.

“Supporting Cell” as used herein in connection with a cochlearepithelium comprises epithelial cells within the organ of Corti that arenot hair cells. This includes inner pillar cells, outer pillar cells,inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells,and/or Claudius cells.

“Synergy” or “synergistic effect” is an effect which is greater than thesum of each of the effects taken separately; a greater than additiveeffect.

“TgfBeta inhibitor” as used herein is a composition that reducesactivity of TgfBeta

“Tissue” is an ensemble of similar cells from the same origin thattogether carry out a specific function including, for example, tissue ofcochlear, such as the Organ of Corti.

“Transtympanic” administration refers to direct injection of acomposition across the tympanic membrane into the middle ear.

“Treating” as used herein in connection with a cell population meansdelivering a substance to the population to effect an outcome. In thecase of in vitro populations, the substance may be directly (or evenindirectly) delivered to the population. In the case of in vivopopulations, the substance may be delivered by administration to thehost subject.

“Valproic acid” (VPA) has chemical formula C₈H₁₆O₂ and the followingalternate name: 2-propylpentanoic acid. Its chemical structure is asfollows:

“Wnt activation” as used herein in connection with a composition is anactivation of the Wnt signaling pathway.

The use of “or” means “and/or” unless stated otherwise. As used in thisapplication, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps. As used in this application,the terms “about” and “approximately” are used as equivalents. Anynumerals used in this application with or without about/approximatelyare meant to cover any normal fluctuations appreciated by one ofordinary skill in the relevant art. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, /toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.For example, inorganic salts include, but are not limited to, ammonium,sodium, potassium, calcium, and magnesium salts. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as ammonia, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Example organic bases used in certain embodiments includeisopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline and caffeine.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions described herein can be formulated in any manner suitablefor a desired delivery route, e.g., transtympanic injection,transtympanic wicks and catheters, and injectable depots. Typically,formulations include all physiologically acceptable compositionsincuding derivatives or prodrugs, solvates, stereoisomers, racemates, ortautomers thereof with any physiologically acceptable carriers,diluents, and/or excipients.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present disclosure relates to compositions, methods, and systems toprevent, reduce or treat the incidence and/or severity of inner eardisorders and hearing impairments involving inner ear tissue,particularly inner ear hair cells, their progenitors, and optionally,the stria vascularis, and associated auditory nerves. Of particularinterest are those conditions that lead to permanent hearing loss wherereduced number of hair cells may be responsible and/or decreased haircell function. Also of interest are those arising as an unwantedside-effect of ototoxic therapeutic drugs including cisplatin and itsanalogs, aminoglycoside antibiotics, salicylate and its analogs, or loopdiuretics. In certain embodiments, the present disclosure relates toinducing, promoting, or enhancing the growth, proliferation orregeneration of inner ear tissue, particularly inner ear supportingcells and hair cells.

Among other things, the compounds presented here are useful for thepreparation of pharmaceutical formulations for the prophylaxis and/ortreatment of acute and chronic ear disease and hearing loss, dizzinessand balance problems especially of sudden hearing loss, acoustic trauma,hearing loss due to chronic noise exposure, presbycusis, trauma duringimplantation of the inner ear prosthesis (insertion trauma), dizzinessdue to diseases of the inner ear area, dizziness related and/or as asymptom of Meniere's disease, vertigo related and/or as a symptom ofMeniere's disease, tinnitus, and hearing loss due to antibiotics andcytostatics and other drugs.

Advantageously, compositions disclosed herein have the capacity toactivate pathways and mechanisms that are known to be involved ininducing stem cell properties, such as those used to create “inducedpluripotent stem cells” (e.g., combined Wnt stimulation, HDACinhibition, TGF-beta inhibition, RAR activation, and/or DKK1suppression). When cochlea supporting cell populations are treated withsuch a composition, whether the population is in vivo or in vitro, thetreated supporting cells exhibit stem-like behavior in that the treatedsupporting cells have the capacity to proliferate and differentiate and,more specifically, differentiate into cochlear hair cells. Preferably,the composition induces and maintains the supporting cells to producedaughter stem cells that can divide for many generations and maintainthe ability to have a high proportion of the resulting cellsdifferentiate into hair cells. In certain embodiments, the proliferatingstem cells express stem cell markers which may include Lgr5, Sox2,Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3,Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tcl1,Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1,Smad2, smad2/3, smad4, smad5, and/or smad7.

In some embodiments, a composition of the present disclosure may be usedto maintain, or even transiently increase stemness (i.e., self-renewal)of a pre-existing supporting cell population prior to significant haircell formation. In some embodiments, the pre-existing supporting cellpopulation comprises inner pillar cells, outer pillar cells, innerphalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and/orClaudius cells. Morphological analyses with immunostaining (includingcell counts) and lineage tracing across a Representative MicroscopySamples may be used to confirm expansion of one or more of thesecell-types. In some embodiments, the pre-existing supporting cellscomprise Lgr5⁺ cells. Morphological analyses with immunostaining(including cell counts) and qPCR and RNA hybridization may be used toconfirm Lgr5 upregulation amongst the cell population.

Advantageously, the methods and compositions of the present disclosureachieve these goals without the use of genetic manipulation. Germ-linemanipulation used in many academic studies is not a therapeuticallydesirable approach to treating hearing loss. In general, the therapypreferably involves the administration of a small molecule, peptide,antibody, or other non-nucleic acid molecule or nucleic acid deliveryvector unaccompanied by gene therapy. In certain embodiments, thetherapy involves the administration of a small organic molecule.Preferably, hearing protection or restoration is achieved through theuse of a (non-genetic) therapeutic that is injected in the middle earand diffuses into the cochlea.

The cochlea relies heavily on all present cell types, and theorganization of these cells is important to their function. Assupporting cells play an important role in neurotransmitter cycling andcochlear mechanics. Thus, maintaining a rosette patterning within theorgan of Corti may be important for function. Cochlear mechanics of thebasilar membrane activate hair cell transduction. Ectopic hair cellshave been created via Atoh1 viral transduction, but it is unlikely thatthese cells can contribute to a hearing response due to theirmislocation in relation to the sensory and mismatch with tectorialmembrane. Furthermore, these cells appear to be more similar tovestibular or non-mammalian hair cells. Thus, more signaling than justAtoh1 or Notch inhibition is necessary for cochlear hair celldevelopment. Due to the high sensitivity of cochlear mechanics, it isalso desirable to avoid masses of cells. In all, maintaining properdistribution and relation of hair cells and supporting cells along thebasilar membrane, even after proliferation, is likely a desired featurefor hearing as supporting cell function and proper mechanics isnecessary for normal hearing.

In a native cochlea, patterning of hair cells and supporting cellsoccurs through Notch lateral inhibition, wherein a cell that becomes ahair cells signals to nearby supporting cells to suppress Atoh1 (a genenecessary for hair cells fate), thereby creating epithelial rosettes. Inone embodiment of the present disclosure, the cell density of hair cellsin a cochlear cell population is expanded in a manner that maintains, oreven establishes, the rosette pattern characteristic of cochlearepithelia.

In accordance with one aspect of the present disclosure, the celldensity of hair cells may be increased in a population of cochlear cellscomprising both hair cells and supporting cells. The cochlear cellpopulation may be an in vivo population (i.e., comprised by the cochlearepithelium of a subject) or the cochlear cell population may be an invitro (ex vivo) population. If the population is an in vitro population,the increase in cell density may be determined by reference to aRepresentative Microscopy Sample of the population taken prior andsubsequent to any treatment. If the population is an in vivo population,the increase in cell density may be determined indirectly by determiningan effect upon the hearing of the subject with an increase in hair celldensity correlating to an improvement in hearing.

In one embodiment, supporting cells placed in a Stem Cell ProliferationAssay in the absence of neuronal cells form ribbon synapses.

In a native cochlea, patterning of hair cells and supporting cellsoccurs in a manner parallel to the basilar membrane. In one embodimentof the present disclosure, the proliferation of supporting cells in acochlear cell population is expanded in a manner that the basilarmembrane characteristic of cochlear epithelia.

In one such embodiment when a composition is applied to cochlear tissue,the number of contiguous hair cells in an expanded cochlear cellpopulation is less than 5% of the hair cells in the population. By wayof further example, in one such embodiment the number of contiguous haircells in an expanded cochlear cell population is less than 4% of thehair cells in the population. By way of further example, in one suchembodiment the number of contiguous hair cells in an expanded cochlearcell population is less than 3% of the hair cells in the population. Byway of further example, in one such embodiment the number of contiguoushair cells in an expanded cochlear cell population is less than 2% ofthe hair cells in the population. By way of further example, in one suchembodiment the number of contiguous hair cells in an expanded cochlearcell population is less than 1% of the hair cells in the population. Insome embodiments, the composition expands inner ear supporting cells togenerate additional hair cells in which the epithelial tissue resemblesNative Morphology which does not have adjacent hair cells for more than5% of hair cells viewed by microscopy

In some embodiments, the composition expands inner ear supporting cellsin animals more than 2 weeks old to generate additional hair cells inwhich the epithelial tissue resembles Native Morphology which does nothave adjacent hair cells for more than 5% of hair cells in aRepresentative Microscopy Sample.

In some embodiments, the composition results in >5% of hair cellscontacting both supporting cells and neurons in a RepresentativeMicroscopy Sample.

In some embodiments, the composition results in hair cells adjacent toother hair cells less than 5% of the time in a Representative MicroscopySample.

In one embodiment, the number of supporting cells in an initial cochlearcell population is selectively expanded by treating the initial cochlearcell population with a composition of the present disclosure (e.g., acomposition containing an Effective Concentration of a Stemness Driverand an Effective Concentration of a Differentiation Inhibitor) to forman intermediate cochlear cell population and wherein the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population. The expanded cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population. For example, in one such embodimentthe ratio of supporting cells to hair cells in the intermediate cochlearcell population exceeds the ratio of supporting cells to hair cells inthe initial cochlear cell population by a factor of 1.1. By way offurther example, in one such embodiment the ratio of supporting cells tohair cells in the intermediate cochlear cell population exceeds theratio of supporting cells to hair cells in the initial cochlear cellpopulation by a factor of 1.5. By way of further example, in one suchembodiment the ratio of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of supportingcells to hair cells in the initial cochlear cell population by a factorof 2. By way of further example, in one such embodiment the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population by a factor of 3. In each of theforegoing embodiments, the capacity of a composition of the presentdisclosure to expand a cochlear cell population as described in thisparagraph may be determined by means of a Stem Cell Proliferation Assay.

In one embodiment, the number of stem cells in a cochlear cellpopulation is expanded to form an intermediate cochlear cell populationby treating a cochlear cell population with a composition of the presentdisclosure (e.g., a composition containing an Effective Concentration ofa Stemness Driver and an Effective Concentration of a DifferentiationInhibitor) wherein the cell density of stem cells in the intermediatecochlear cell population exceeds the cell density of stem cells in theinitial cochlear cell population. The treated cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the cell density ofstem cells in the treated cochlear cell population exceeds the celldensity of stem cells in the initial cochlear cell population by afactor of at least 1.1. For example, in one such embodiment the celldensity of stem cells in the treated cochlear cell population exceedsthe cell density of stem cells in the initial cochlear cell populationby a factor of at least 1.25. For example, in one such embodiment thecell density of stem cells in the treated cochlear cell populationexceeds the cell density of stem cells in the initial cochlear cellpopulation by a factor of at least 1.5. By way of further example, inone such embodiment the cell density of stem cells in the treatedcochlear cell population exceeds the cell density of stem cells in theinitial cochlear cell population by a factor of at least 2. By way offurther example, in one such embodiment the cell density of stem cellsin the treated cochlear cell population exceeds the cell density of stemcells in the initial cochlear cell population by a factor of at least 3.In vitro cochlear cell populations may expand significantly more than invivo populations; for example, in certain embodiments the cell densityof stem cells in an expanded in vitro population of stem cells may be atleast 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2,000 or even 3,000 timesgreater than the cell density of the stem cells in the initial cochlearcell population. In each of the foregoing embodiments, the capacity of acomposition of the present disclosure to expand a cochlear cellpopulation as described in this paragraph may be determined by means ofa Stem Cell Proliferation Assay.

In accordance with one aspect of the present disclosure, a cochleasupporting cell population is treated with a composition of the presentdisclosure (e.g., a composition containing an Effective Concentration ofa Stemness Driver and an Effective Concentration of a DifferentiationInhibitor) to increase the Lgr5 activity of the population. For example,in one embodiment the composition has the capacity to increase andmaintain the Lgr5 activity of an in vitro population of cochleasupporting cells by factor of at least 1.2. By way of further example,in one such embodiment the composition has the capacity to increase theLgr5 activity of an in vitro population of cochlea supporting cells byfactor of 1.5. By way of further example, in one such embodiment thecomposition has the capacity to increase the Lgr5 activity of an invitro population of cochlea supporting cells by factor of 2, 3, 5 10,100, 500, 1,000, 2,000 or even 3,000. Increases in Lgr5 activity mayalso be observed for in vivo populations but the observed increase maybe somewhat more modest. For example, in one embodiment the compositionhas the capacity to increase the Lgr5 activity of an in vivo populationof cochlea supporting cells by at least 5%. By way of further example,in one such embodiment the composition has the capacity to increase theLgr5 activity of an in vivo population of cochlea supporting cells by atleast 10%. By way of further example, in one such embodiment thecomposition has the capacity to increase the Lgr5 activity of an in vivopopulation of cochlea supporting cells by at least 20%. By way offurther example, in one such embodiment the composition has the capacityto increase the Lgr5 activity of an in vivo population of cochleasupporting cells by at least 30%. In each of the foregoing embodiments,the capacity of the composition for such an increase in Lgr5 activitymay be demonstrated, for example, in an In Vitro Lgr5⁺ Activity Assayand in an in vivo population may be demonstrated, for example, in an InVivo Lgr5⁺ Activity Assay, as measured by isolating the organ andperforming morphological analyses using immunostaining, endogenousfluorescent protein expression of Lgr5 (eg. Lgr5, Sox2), and qPCR forLgr5.

In addition to increasing the Lgr5 activity of the population, thenumber of Lgr5⁺ supporting cells in a cochlea cell population may beincreased by treating a cochlea cell population containing Lgr5⁺supporting cells (whether in vivo or in vitro) with a composition of thepresent disclosure (e.g., a composition containing an EffectiveConcentration of a Sternness Driver and an Effective Concentration of aDifferentiation Inhibitor). In general, the cell density of thestem/progenitor supporting cells may expand relative to the initial cellpopulation via one or more of several mechanisms. For example, in onesuch embodiment, newly generated Lgr5⁺ supporting cells may be generatedthat have increased stem cell propensity (i.e., greater capacity todifferentiate into hair cell). By way of further example, in one suchembodiment no daughter Lgr5⁺ cells are generated by cell division, butpre-existing Lgr5⁺ supporting cells are induced to differentiate intohair cells. By way of further example, in one such embodiment nodaughter cells are generated by cell division, but Lgr5⁻ supportingcells are activated to a greater level of Lgr5 activity and theactivated supporting cells are then able to differentiate into haircells. Regardless of the mechanism, in one embodiment a composition ofthe present disclosure has the capacity to increase the cell density ofLgr5⁺ supporting cells in an in vitro isolated cell population ofcochlea supporting cells by factor of at least 5. By way of furtherexample, in one such embodiment the composition has the capacity toincrease the cell density of Lgr5⁺ supporting cells in an in vitropopulation of cochlea supporting cells by factor of at least 10. By wayof further example, in one such embodiment the composition has thecapacity to increase the cell density of Lgr5⁺ supporting cells in an invitro population of cochlea supporting cells by factor of at least 100,at least 500, at least 1,000 or even at least 2,000. Increases in thecell density of Lgr5⁺ supporting cells may also be observed for in vivopopulations but the observed increase may be somewhat more modest. Forexample, in one embodiment the composition has the capacity to increasethe cell density of Lgr5⁺ supporting cells in an in vivo population ofcochlea supporting cells by at least 5%. By way of further example, inone such embodiment the composition has the capacity to increase thecell density of Lgr5⁺ supporting cells in an in vivo population ofcochlea supporting cells by at least 10%. By way of further example, inone such embodiment the composition has the capacity to increase thecell density of Lgr5⁺ supporting cells in an in vivo population ofcochleasupporting cells by at least 20%. By way of further example, inone such embodiment the composition has the capacity to increase thecell density of Lgr5⁺ supporting cells in an in vivo population ofcochlea supporting cells by at least 30%. The capacity of thecomposition for such an increase in Lgr5⁺ supporting cells in an invitro population may be demonstrated, for example, in a Stem CellProliferation Assay or in an appropriate in vivo assay. In oneembodiment, a composition of the present disclosure has the capacity toincrease the number of Lgr5⁺ cells in the cochlea by inducing expressionof Lgr5 in cells with absent or low detection levels of the protein,while maintaining Native Morphology. In one embodiment, a composition ofthe present disclosure has the capacity to increase the number of Lgr5⁺cells in the cochlea by inducing expression of Lgr5 in cells with absentor low detection levels of the protein, while maintaining NativeMorphology and without producing Cell Aggregates.

In addition to increasing the cell density of Lgr5⁺ supporting cells, inone embodiment a composition of the present disclosure has the capacityto increase the ratio of Lgr5⁺ cells to hair cells in a cochlear cellpopulation. In one embodiment, the number of Lgr5⁺ supporting cells inan initial cochlear cell population is selectively expanded by treatingthe initial cochlear cell population with a composition of the presentdisclosure (e.g., a composition containing an Effective Concentration ofa Stemness Driver and an Effective Concentration of a DifferentiationInhibitor) to form an expanded cell population and wherein the number ofLgr5⁺ supporting cells in the expanded cochlear cell population at leastequals the number of hair cells. The expanded cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1:1. For example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1.5:1. Byway of further example, in one such embodiment theratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlearcell population is at least 2:1. By way of further example, in one suchembodiment the ratio of Lgr5⁺ supporting cells to hair cells in theexpanded cochlear cell population is at least 3:1. By way of furtherexample, in one such embodiment the ratio of Lgr5⁺ supporting cells tohair cells in the expanded cochlear cell population is at least 4:1. Byway of further example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 5:1. In each of the foregoing embodiments, the capacity of acomposition of the present disclosure to expand a cochlear cellpopulation as described in this paragraph may be determined by means ofa Stem Cell Proliferation Assay.

In certain embodiments, the composition increases the fraction of theLgr5⁺ cells to total cells on the sensory epithelium by at least 10%,20%, 50%, 100%, 250% 500%, 1,000% or 5000%.

In certain embodiments, the composition increases the Lgr5⁺ cells untilthey become at least 10, 20, 30, 50, 70, or 85% of the cells on thesensory epithelium, e.g. the Organ of Corti.

In general, excessive proliferation of supporting cells in the cochleais preferably avoided. In one embodiment, a composition of the presentdisclosure has the capacity to expand a cochlear cell population withoutcreating a protrusion of new cells beyond the native surface of thecochlea, e.g a Cell Aggregate. In some embodiments, 30 days afterplacing a composition on the round window membrane, the cochlear tissuehas Native Morphology. In some embodiments, 30 days after placing acomposition on the round window membrane, the cochlear tissue has NativeMorphology and lacks Cell Aggregates. In some embodiments, 30 days afterplacing a composition on the round window membrane, the cochlear tissuehas Native Morphology and at least 10, 20, 30, 50, 75, 90, 95, 98, oreven at least 99% of the Lgr5⁺ cells in the Organ of Corti are not partof Cell Aggregates.

In addition to expanding supporting cell populations, generally, andLgr5⁺ supporting cells, specifically, as described above, compositionsof the present disclosure (e.g., a composition containing an EffectiveConcentration of a Stemness Driver and an Effective Concentration of aDifferentiation Inhibitor) have the capacity to maintain, in thedaughter cells, the capacity to differentiate into hair cells. In invivo populations, the maintenance of this capacity may be indirectlyobserved by an improvement in a subject's hearing. In in vitropopulations, the maintenance of this capacity may be directly observedby an increase in the number of hair cells relative to a startingpopulation or indirectly by measuring LGR5 activity, SOX2 activity orone or more of the other stem cell markers identified elsewhere herein.

In one embodiment, the capacity of a composition to increase thestemness of a population of cochlear supporting cells, in general, or apopulation of Lgr5⁺ supporting cells, in particular, may be correlatedwith an increase of Lgr5 activity of an in vitro population of isolatedLgr5⁺ cells as determined by an Lgr5 Activity Assay. As previouslynoted, in one such embodiment, the composition has the capacity toincrease the Lgr5 activity of stem cells in the intermediate cellpopulation by a factor of 5 on average relative to the Lgr5 activity ofthe cells in the initial cell population. By way of further example, inone such embodiment the composition has the capacity to increase theLgr5 activity of the stem cells genes in the intermediate cellpopulation by a factor of 10 relative to the Lgr5 activity of the cellsin the initial cell population. By way of further example, in one suchembodiment the composition has the capacity to increase the Lgr5activity of the stem cells in the intermediate cell population by afactor of 100 relative to the Lgr5 activity of the cells in the initialcell population. By way of further example, in one such embodiment thecomposition has the capacity to increase the Lgr5 activity of the stemcells in the intermediate cell population by a factor of 1000 relativeto the Lgr5 activity of the cells in the initial cell population. Ineach of the foregoing embodiments, the increase in the activity of stemcells in the cell population may be determined in vitro byimmunostaining or endogenous fluorescent protein expression for targetgenes and analysis of their relative intensities via imaging analysis orflowcytometry, or using qPCR for target stem cell genes. The identity ofthe resulting stem cell population may optionally be further determinedby stem cell assays including stem cell marker expression assay, colonyforming assay, self-renewal assay and differentiation assay as definedin Stem cell assay.

In some embodiments, the method applied to an adult mammal produces apopulation of adult mammalian Lgr5⁺ cells that are in S-phase.

In one embodiment, after applying a composition to the round window of amouse, the in vivo Lgr5⁺ Activity of a cell population in the Organ ofCorti increases 1.3×, 1.5×, up to 20× over baseline for a populationthat has not been exposed to the composition. In some embodiments,applying a composition to the round window of a mouse increases theaverage In vivo Lgr5⁺ Activity for cells in the Organ of Corti isincreased 1.3×, 1.5×, up to 20× over baseline for a population that hasnot been exposed to the composition.

In certain embodiments, the composition increases the Lgr5⁺ cells untilthey become at least 10%, 7.5%, 10%, up to 100% of the supporting cellpopulation by number.

In some cases, a Stemness Driver may also induce differentiation of thesupporting cells to hair cells if a Differentiation Inhibitor is notpresent at an Effective Differentiation Inhibition Concentration.Examples of Stemness Drivers that may drive both proliferation anddifferentiation include GSK3Beta inhibitors and Wnt agonists. In certainembodiments, the proliferation of the stem cells may be enhanced byadding a modulator of pathways that regulate cell cycle or plasticity,such as the p27 or TgfBeta pathways.

In some embodiments, a Stemness Driver may be used to drive theproliferation of Lgr5⁺ stem cells. In some cases, a Stemness Driver mayalso induce differentiation of Lgr5⁺ cells to hair cells if aDifferentiation Inhibitor is not present at an Effective DifferentiationInhibition Concentration. Examples of Stemness Drivers that may driveboth proliferation and differentiation include GSK3Beta inhibitors andWnt agonists. In some embodiments, the Differentiation inhibitor is alsoa Stemness Driver. In some embodiments, the Differentiation inhibitor isa Notch agonist and is also a Stemness Driver. In some embodiments, theDifferentiation inhibitor is Valproic Acid, which may be a StemnessDriver. If a Differentiation Inhibitor is also a Stemness Driver, theconcentration of the Differentiation Inhibitor should fall below theEffective Differentiation Inhibition Concentration during theDifferentiation Period.

In certain embodiments, a combination of (i) a GSK3-beta inhibitorand/or Wnt agonist, and (ii) a notch agonist and/or HDAC inhibitor isused, which has the capacity to increase stem cell population by atleast 3 times larger than the stem cell population prior to theadministering step when applied to Lgr5⁺ cells obtained from inner earof mice.

In certain embodiments, a composition has the capacity to increase thepercentage of Lgr5⁺ cell in a cochlea by 5%, 10%, 25%, 50%, or 80%. Incertain embodiments, a combination of (i) a GSK3-beta inhibitor and/orWnt agonist, and (ii) a notch agonist and/or HDAC inhibitor is used,which has the capacity to increase the percentage of Lgr5⁺ cell in acochlea by 5%, 10%, 25%, 50%, or 80%.

Stemness Drivers

Exemplary GSK3-beta inhibitors within the present disclosure appear inTable 1.

TABLE 1 GSK3β Inhibitors Column A Column B Class Agent AloisinesAloisine A Aloisines Aloisine B Aminopyridine CT20026 AminopyrimidineCHIR99021 (CT99021) Aminopyrimidine CHIR98014 (CT98014) AminopyrimidineCHIR98023 (CT98023) Aminopyrimidine TWS119 Anilinoarylmaleimide I5Arylindolemaleimide SB-216763 Arylindolemaleimide SB-415286 (SB-41528)Azaindolylmaleimide Compound 29 Azaindolylmaleimide Compound 46Benzazepinone Kenpaullone Benzazepinone Alsterpaullone BenzazepinoneAzakenpaullone Bis-Indole Indirubin-30-oxime Bis-Indole6-Bromoindirubin-30-oxime (BIO) Bis-Indole 6-Bromoindirubin-30-acetoximeBisindolylmaleimide Staurosporine Bisindolylmaleimide Compound 5aBisindolylmaleimide GF109203x (bisindolylmaleimide I)Bisindolylmaleimide Ro318220 (bisindolylmaleimide IX)Bisindolylmaleimide Bisindolylmaleimide × hydrochlorideBisindolylmaleimide Enzastaurin Chloromethyl thienyl ketone Compound 17Flavone Flavopiridol Furanosesquiterpenes PalinurineFuranosesquiterpenes Tricantine Halomethylketones HMK-32 IndirubinsIndirubin-3′-monoxime Indirubins 6-BIO Indirubins5-lodo-indirubin-3′-monoxime Indirubins Indirubin-5-sulfonic acid sodiumsalt Inorganic atom Lithium Inorganic atom Beryllium Inorganic atom ZincInorganic atom Tungstate Manzamines Manzamine A Oxindole SU9516Organometallic HB12 Organometallic DW12 Organometallic NP309Organometallic (RRu)-HB1229 Organometallic (RRu)-NP549 OrganometallicGSK3 inhibitor XV Paullones Kenpaullone Paullones AlsterpaullonePaullones Cazpaullone Paullones 9-Cyanopaullone Peptide FRATtide PeptideL803 Peptides L803-mts Phenylaminopyrimidine CGP60474 PyrazolopyridinePyrazolopyridine 9 Pyrazolopyridine Pyrazolopyridine 18 PyrazolopyridinePyrazolopyridine 34 Pyrazolopyrimidine Compound 1A PyrazoloquinoxalineCompound 1 Pyridyloxadiazole Compound 12 Pyrroloazepine HymenialdisinePyrrolopyrazine Aloisine A Pyrrolopyrazine Aloisine B PyrrolopyrimidineTWS119 Thiadiazolidindiones TDZD-8 Thiadiazolidindiones NP00111Thiadiazolidindiones NP031115 Thiadiazolidindiones NP031112 (Tideglusib)Thiazoles AR-A014418 Thiazoles AZD-1080 Triazole Compound 8bMiscellaneous Hymenialdisine Miscellaneous DibromocantharellineMiscellaneous KT 5720 Miscellaneous CID 755673 Miscellaneous GSK-3βInhibitor VII Miscellaneous Hymenidin Miscellaneous 3F8 MiscellaneousTCS 2002 Miscellaneous TCS 21311 Miscellaneous A 1070722 MiscellaneousTC-G 24 Miscellaneous Bikinin Miscellaneous LY2090314 Miscellaneous10Z-Hymenialdisine Miscellaneous NSC 693868 Miscellaneous IM-12Miscellaneous Indirubin Miscellaneous AZD2858 (AR28) MiscellaneousGSK-3β Inhibitor I Miscellaneous GSK-3 Inhibitor II Miscellaneous GSK-3βInhibitor VIII Miscellaneous GSK-3 Inhibitor XXII MiscellaneousIndirubin-3′-monoxime-5-sulphonic Acid Miscellaneous GSK-3 InhibitorXIII Miscellaneous GSK3β Inhibitor XIX Miscellaneous GSK-3β InhibitorXXVII Miscellaneous GSK-3beta Inhibitor XXVI Miscellaneous GSK-3βInhibitor XII Miscellaneous GSK-3β Inhibitor XXIV Miscellaneous GSK-3Inhibitor XV Miscellaneous GSK-3β Inhibitor VI Miscellaneous GSK-3βInhibitor XVIII Miscellaneous GSK-3 Inhibitor XXIX Miscellaneous GSK-3Inhibitor IV Miscellaneous GSK-3β Inhibitor VII Miscellaneous GSK-3Inhibitor IX Miscellaneous GSK-3 Inhibitor X Miscellaneous GSK-3βInhibitor XXV Miscellaneous GSK-3 Inhibitor XVI Miscellaneous GSK-3βInhibitor XI Miscellaneous GSK-3 inhibitor 1 Miscellaneous A-1070722Miscellaneous 3-Amino-1H-pyrazolo[3,4-b]quinoxaline Miscellaneous GSK3Inhibitor, 2 Miscellaneous GSK-3beta Inhibitor III Miscellaneous AR-AO14418-d3 Miscellaneous ML320 Miscellaneous BIP-135 Miscellaneous CP21R7Publication NP-103 Publication CG-301338 Publication SAR 502250Publication XD-4241 Publication CEP-16805 Lithium Chloride and otherinorganic atoms. Lithium, Beryllium, Zinc, Tungstate MaleimideDerivatives such as SB-216763 e.g. Compound 1-30, SB-216763, etcStaurosporine and Organometallic inhibitors e.g. Compound 31-37, etcIndole Derivatives such as Indirubin-3′-monoxime e.g. Compound 38-50,BIO, etc and BIO Paullone Derivatives such as Kenpallone and e.g.Compound 51-64, Kenpaullone, etc Alsterpaullone Pyrazolamide derivativese.g. Compound 65-78, etc Pyrimidine and Furopyrimidine derivatives suchas e.g. Compound 79-102, CHIR99021, CHIR98014, CHIR99021 and CHIR98014etc Oxadiazole derivatives e.g. Compound 103-127, etc Thiazolederivatives such as AR-A014418 e.g. Compound 128-130, AR-A01448, etcMiscellaneous Heterocyclic derivative Compound 131-154 Publication AR79Publication AZ13282107 Publication AR28 (AZD2858) Patent GI179186XPatent CT118637 Patent CP-70949 Patent GW784752X Patent GW784775XPublication CT73911 Publication CT20026 Publication LY2064827Publication 705701 Publication 708244 Publication 709125 Patent WO2008077138 A1 Patent WO 2003037891 A1 Patent U.S. Pat. No. 8,207,216 B2Patent U.S. Pat. No. 8,071,591 B2 Patent CN 1319968 C Patent U.S. Pat.No. 7,514,445 B2 Patent CN 101341138 B Patent EP 1961748 A2 Patent WO2010104205 A1 Patent U.S. 20100292205 A1 Patent WO 2014003098 A1 PatentWO 2011089416 A1 Patent EP 1739087 A1 Patent WO 2001085685 A1 PatentU.S. 20070088080 A1 Patent WO 2006018633 A1 Patent WO 2009017453 A1Patent WO 2014050779 A1 Patent W02006100490A1/EP 1863904 A1 Patent WO2014013255 A1 Patent WO 2009017455 A1 Patent EP 2765188 A1 Patent WO2014083132 A1 Patent U.S. Pat. No. 8,771,754 B2 Patent WO 2013124413 A1Patent WO 2014059383 A1 Patent WO 2010075551 A1 Patent U.S. Pat. No.8,686,042 B2 Patent WO 2007102770 A1 Aminopyrimidine CHIR99021 (CT99021)Miscellaneous LY2090314 Aminopyrimidine TWS119 ArylindolemaleimideSB-216763 Benzazepinone Azakenpaullone Paullones CazpaullonePyrazolopyridine Pyrazolopyridine 9 Miscellaneous TCS 2002 MiscellaneousA 1070722 Aminopyrimidine CHIR98014 (CT98014) Benzazepinone KenpaulloneInorganic atom Lithium Miscellaneous ML320 Miscellaneous CP21R7

Classes of GSK3-beta inhibitors for use in various embodiments of thecompositions and methods disclosed herein include but are not limited tothose listed in Column A of Table 1. Specific GSK3-beta inhibitors foruse in various embodiments of the compositions and methods disclosedherein include but are not limited to those listed in Column B ofTable 1. Classes of Wnt agonists for use in various embodiments of thecompositions and methods disclosed herein include but are not limited tothose listed in Column A of Table 2. Specific Wnt agonists for use invarious embodiments of the compositions and methods disclosed hereininclude but are not limited to those listed in Column B of Table 2.Classes of notch agonists for use in various embodiments of thecompositions and methods disclosed herein include but are not limited tothose listed in Column A of Table 3. Specific notch agonists for use invarious embodiments of the compositions and methods disclosed hereininclude but are not limited to those listed in Column B of Table 3.Classes of HDAC inhibitors for use in various embodiments of thecompositions and methods disclosed herein include but are not limited tothose listed in Column A of Table 4. Specific HDAC inhibitors for use invarious embodiments of the compositions and methods disclosed hereininclude but are not limited to those listed in Column B of Table 4. Allagents listed in Table 1 column B, Table 2 column B, Table 3 column B,Table 4 column B are understood to include derivatives orpharmaceutically acceptable salts thereof. All classes listed in Table 1column A, Table 2 column A, Table 3 column A, Table 4 column A areunderstood to include both agents comprising that class and derivativesor pharmaceutically acceptable salts thereof. Members of each of theseclasses also include but are not limited to those described in pages51-55 of Appendix A and pages 90-102 or Appendix A.

GSK3-beta inhibitors also include but are not limited to those agentsthat reduce GSK3-beta activity by more than 5, 10, 20, 30, or 50% whenan otic cell line or primary cells obtained from otic tissue is exposedto the inhibitor at pharmaceutically acceptable concentrations andactivity is assessed via Western blotting or other standard methods inthe literature. A pharmaceutically acceptable concentration, as usedherein, is a concentration of an active agent in a formulation that isnon-toxic and can be delivered to the tissue of interest. In certainembodiments, the composition comprises a GSK3-beta inhibitor reducingGSK3-beta activity by more than 5, 10, 20, 30, or 50% using conditionsdescribed in this paragraph in combination with a notch agonist and/orHDAC inhibitor. “Highly potent GSK3-beta inhibitors are those thatreduce GSK3-beta activity by more than 50% when an otic cell line orprimary cells obtained from otic tissue is exposed to the inhibitor atpharmaceutically acceptable concentrations and activity is assessed viaWestern blotting or other standard methods in the literature.

Wnt Agonists

Exemplary Wnt agonists within the present disclosure appear in Table 2.

TABLE 2 Wnt Agonist Column A Column B Wnt Ligand Wnt1/Int-1 Wnt-2/Irp(Int-I-related protein) Wnt-2b/13 Wnt-3/Int-4 Wnt-3a Wnt-4 Wnt-5a Wnt-5bWnt-6 Wnt-7a Wnt-7b Wnt-8a/8d Wnt-8b Wnt-9a/14 Wnt-9b/14b/15 Wnt-10aWnt-10b/12 Wnt-11 Wnt-16 Wnt Related Protein R-Spondin 1/2/3/4 NorrinGSK3b inhibitor Other Wnt modulator (hetero)arylpyrimidines Wnt AgonistIQ 1 DCA QS 11 WASP-1, ZINC00087877 WAY 316606, HCl WAY-262611, HClHLY78 SKL2001 Cpd1 Cpd2 cmpd 109 ISX 9 Cmpd 71 Cmpd 2 Selumetinib(AZD6244) Radicicol (hetero)arylpyrimidines Wnt Agonist WAY 316606, HClWAY-262611, HCl SKL2001 ISX 9

Wnt-agonists also include but are not limited to those agents thatincrease Wnt activity by more than 5, 10, 20, 30, or 50% when an oticcell line or primary cells obtained from otic tissue is exposed to theagonist at pharmaceutically acceptable concentrations and activity isassessed via Western blotting or other standard methods in theliterature. In certain embodiments, the composition comprises a Wntagonist increasing Wnt activity by more than 5, 10, 20, 30, or 50% usingconditions described in this paragraph in combination with a notchagonist and/or HDAC inhibitor. “Highly potent Wnt agonist are those thatincrease Wnt activity by more than 50% when an otic cell line or primarycells obtained from otic tissue is exposed to the agonist atpharmaceutically acceptable concentrations and activity is assessed viaWestern blotting or other standard methods in the literature.

Differentiation Inhibitors Notch Agonists

Exemplary Notch agonists within the present disclosure appear in Table3.

TABLE 3 Notch Agonist Column A Column B Natural receptor Ligands Jagged1 Jagged 2 Delta-like 1 Delta-like 2 Delta-like 3 Delta-like 4 DSLpeptide Delta 1 Delta D Receptor antibodies Notch 1 antibody HDACinhibitors VPA TSA Tubastatin A Compound 7

Notch-agonistsalsoincludebutarenotlimitedtothoseagentsthatincreaseNotchactivity by more than 5, 10, 20, 30, or 5000 when an otic cell line orprimary cells obtained from otic tissue is exposed to the agonist atpharmaceutically acceptable concentrations and activity is assessed viaWestern blotting or other standard methods in the literature. In certainembodiments, the composition comprises a Notch agonist increasing Notchactivity by more than 5, 10, 20, 30, or 50% using conditions describedin this paragraph in combination with a GSK3-beta inhibitor or Wntagonist. “Highly potent Notch agonist are those that increase Notchactivity by more than 50% when an otic cell line or primary cellsobtained from otic tissue is exposed to the agonist at pharmaceuticallyacceptable concentrations and activity is assessed via Western blottingor other standard methods in the literature.

HDAC Inhibitors

Exemplary HDAC Inhibitors within the present disclosure appear in Table4.

TABLE 4 HDAC Inhibitors Column A Column B Class Agent HydroxamatesTrichostatin A (TSA) Hydroxamates SAHA (Zolinza, vorinostat)Hydroxamates 4-iodo-SAHA Hydroxamates SBHA Hydroxamates CBHAHydroxamates LAQ-824 Hydroxamates PDX-101 (belinostat) HydroxamatesLBH-589 (panobinostat) Hydroxamates ITF2357 (Givinostat) HydroxamatesPCI-34051 Hydroxamates PCI-24781 (Abexinostat) Hydroxamates Tubastatin AHydroxamates CUDC-101 Hydroxamates Compound 7 Hydroxamates OxamflatinHydroxamates ITF2357 Hydroxamates Bufexamac Hydroxamates APHA Compound 8Hydroxamates JNJ-26481585 (Quisinostat) Hydroxamates Suberoylanilide-d5Hydroxamic Acid Hydroxamates HDAC Inhibitor XXIV Hydroxamates TubacinHydroxamates Butyrylhydroxamic acid Hydroxamates 1-NaphthohydroxamicAcid Hydroxamates MC 1568 Hydroxamates SB939 (Pracinostat) Hydroxamates4SC-201 (Resminostat) Hydroxamates Tefinostat (CHR-2845) HydroxamatesCHR-3996 Hydroxamates CG200745 Cyclic peptide Depsipeptide (Romidepsin,FK-228, FR 901228) Cyclic peptide Trapoxin A Cyclic peptide HC ToxinAliphatic Acid Valproic Acid Aliphatic Acid VAHA Aliphatic Acid Phenylbutyrate Aliphatic Acid Butyrate Aliphatic Acid AN-9 Benzamides MS-275(Entinostat) Benzamides MGCD0103 (Mocetinostat) Benzamides CI994(Tacedinaline; PD-123654; GOE-5549; Acetyldinaline) Benzamides BML-210Hydroxamates M 344 Benzamides Chidamide Hydroxamates4-(dimethylamino)-N-[6-(hydroxyamino)-6- oxohexyl]-benzamideMiscellaneous Luteolin Prodrug of thiol PTACH Miscellaneous L-CarnitineMiscellaneous Biphenyl-4-sulfonyl chloride Miscellaneous SIRT1/2Inhibitor VII Hydroxamates (S)-HDAC-42 Hydroxamates Indole-3-acetamideMiscellaneous NSC 3852 Miscellaneous PPM-18 Miscellaneous Ratjadone A,Synthetic Benzamides N-(2-Aminophenyl)-N′-phenylheptanediamideMiscellaneous Dihydrochlamydocin Miscellaneous 7-AminoindoleMiscellaneous Apicidin Miscellaneous Parthenolide Hydroxamates HNHAMiscellaneous Splitomicin Benzamides RGFP109 Benzamides RGFP136Benzamides RGFP966 Benzamides 4SC-202 Hydroxamates ACY1215 MiscellaneousME-344 Miscellaneous Sulforaphane CF3Methyl Ketones 6H CF3Methyl Ketones27 Aryl Ketones 25 Non classical 5 Nexturastat A Droxinostat AR-42Romidepsin (FK228, Depsipeptide) Scriptaid Sodium Phenylbutyrate TMP269Thailandepsin A BRD9757 LMK235 HPOB CAY10603 Tasquinimod HDAC6 InhibitorIII HDAC Inhibitor XXIV HDAC Inhibitor IV HDAC Inhibitor XIX HDACInhibitor XXII HDAC Inhibitor VII HDAC Inhibitor II HDAC Inhibitor VI(−)-Depudecin KD 5170 TC-H 106 TCS HDAC6 20b Pyroxamide ChidamideHDAC-IN-1 HC Toxin Hydroxamates SAHA (Zolinza, vorinostat) HydroxamatesLBH-589 (panobinostat) Hydroxamates JNJ-26481585 (Quisinostat) Cyclicpeptide Depsipeptide (Romidepsin, FK-228, FR 901228) Benzamides MGCD0103(mocetinostat) Prodrug of thiol PTACH Miscellaneous Ratjadone A,Synthetic Miscellaneous Apicidin CF3Methyl Ketones 27 Non classical 5Nexturastat A Droxinostat Scriptaid BRD9757 HPOB CAY10603 HDAC6Inhibitor III Hydroxamates ACY1215 Hydroxamates Tubastatin AHydroxamates Tubacin Hydroxamates Trichostatin A (TSA)

HDAC inhibitors also include but are not limited to those agents thatreduce HDAC activity by more than 5, 10, 20, 30, or 50% when an oticcell line or primary cells obtained from otic tissue is exposed to theinhibitor and activity is assessed via Western blotting or otherstandard methods in the literature. In certain embodiments, thecomposition comprises a HDAC inhibitor decreasing HDAC activity by morethan 5, 10, 20, 30, or 50% using conditions described in this paragraphin combination with a GSK3-beta inhibitor or Wnt agonist. “Highly potentHDAC agonist are those that decreases HDAC activity by more than 50%when an otic cell line or primary cells obtained from otic tissue isexposed to the inhibitor at pharmaceutically acceptable concentrationsand activity is assessed via Western blotting or other standard methodsin the literature.

Representative methods to assess HDAC inhibitors may be found in:

-   Mak B C, Takemaru K, Kenerson H L, Moon R T, Yeung R S (2003). “The    tuberin-hamartin complex negatively regulates beta-catenin signaling    activity”. J. Biol. Chem. 278(8): 5947-51.    doi:10.1074/jbc.C200473200. PMID 12511557.-   Nakamura T, Hamada F, Ishidate T, Anai K, Kawahara K, Toyoshima K,    Akiyama T (1998). “Axin, an inhibitor of the Wnt signalling pathway,    interacts with beta-catenin, GSK-3beta and APC and reduces the    beta-catenin level”. Genes Cells 3 (6):    395-403.doi:10.1046/j.1365-2443.1998.00198.x. PMID 9734785.-   von Kries J P, Winbeck G, Asbrand C, Schwarz-Romond T, Sochnikova N,    Dell'Oro A, Behrens J, Birchmeier W (2000). “Hot spots in    beta-catenin for interactions with LEF-1, conductin and APC”. Nat.    Struct. Biol. 7 (9): 800-7. doi:10.1038/79039.PMID 10966653.-   Schwarz-Romond T, Asbrand C, Bakkers J, Kühl M, Schaeffer H J,    Huelsken J, Behrens J, Hammerschmidt M, Birchmeier W (2002). “The    ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to    the beta-catenin degradation complex and acts in both canonical Wnt    and Wnt/JNK signaling”. Genes Dev. 16 (16): 2073-84.    doi:10.1101/gad.230402.PMC 186448. PMID 12183362.-   Wang L, Lin H K, Hu Y C, Xie S, Yang L, Chang C (2004). “Suppression    of androgen receptor-mediated transactivation and cell growth by the    glycogen synthase kinase 3 beta in prostate cells”. J. Biol. Chem.    279 (31): 32444-52. doi:10.1074/jbc.M313963200.PMID 15178691.-   Davies G, Jiang W G, Mason M D (2001). “The interaction between    beta-catenin, GSK3beta and APC after motogen induced cell-cell    dissociation, and their involvement in signal transduction pathways    in prostate cancer”. Int. J. Oncol. 18 (4):    843-7.doi:10.3892/ijo.18.4.843. PMID 11251183.-   Kishida S, Yamamoto H, Hino S, Ikeda S, Kishida M, Kikuchi A (1999).    “DIX domains of Dvl and axin are necessary for protein interactions    and their ability to regulate beta-catenin stability”. Mol. Cell.    Biol. 19 (6): 4414-22. PMC 104400. PMID 10330181.-   Hong Y R, Chen C H, Cheng D S, Howng S L, Chow C C (1998). “Human    dynamin-like protein interacts with the glycogen synthase kinase    3beta”. Biochem. Biophys. Res. Commun. 249 (3): 697-703.    doi:10.1006/bbrc.1998.9253. PMID 9731200.-   Wu, Xiaoyang; Shen Q T; Oristian D S; Lu C P; Zheng Q; Wang H W;    Fuchs E (2011). “Skin Stem Cells Orchestrate Directional Migration    by Regulating Microtubule-ACF7 Connections through GSK3b”. Cell 144.    doi:10.1016/j.cell.2010.12.033.-   Li Y, Bharti A, Chen D, Gong J, Kufe D (1998). “Interaction of    glycogen synthase kinase 3beta with the DF3/MUC1    carcinoma-associated antigen and beta-catenin”. Mol. Cell. Biol. 18    (12): 7216-24. PMC 109303. PMID 9819408.-   Li Y, Kuwahara H, Ren J, Wen G, Kufe D (2001). “The c-Src tyrosine    kinase regulates signaling of the human DF3/MIUC1    carcinoma-associated antigen with GSK3 beta and beta-catenin”. J.    Biol. Chem. 276 (9): 6061-4. doi:10.1074/jbc.C000754200.PMID    11152665.-   Guo X, Ramirez A, Waddell D S, Li Z, Liu X, Wang X F (2008). “Axin    and GSK3-control Smad3 protein stability and modulate    TGF-signaling”. Genes Dev. 22 (1): 106-20.doi:10.1101/gad.1590908.    PMC 2151009. PMID 18172167.-   Foltz D R, Santiago M C, Berechid B E, Nye J S (2002). “Glycogen    synthase kinase-3beta modulates notch signaling and stability”.    Curr. Biol. 12 (12): 1006-11. doi:10.1016/50960-9822(02)00888-6.    PMID 12123574.-   Espinosa L, Ingles-Esteve J, Aguilera C, Bigas A (2003).    “Phosphorylation by glycogen synthase kinase-3 beta down-regulates    Notch activity, a link for Notch and Wnt pathways”. J. Biol. Chem.    278 (34): 32227-35. doi:10.1074/jbc.M304001200. PMID 12794074.-   Watcharasit P, Bijur G N, Zmijewski J W, Song L, Zmijewska A, Chen    X, Johnson G V, Jope R S (2002). “Direct, activating interaction    between glycogen synthase kinase-3beta and p53 after DNA damage”.    Proc. Natl. Acad. Sci. U.S.A. 99 (12):    7951-5.doi:10.1073/pnas.122062299. PMC 123001. PMID 12048243.-   Dai F, Yu L, He H, Chen Y, Yu J, Yang Y, Xu Y, Ling W, Zhao S    (2002). “Human serum and glucocorticoid-inducible kinase-like kinase    (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta)    at serine-9 through direct interaction”. Biochem. Biophys. Res.    Commun. 293 (4): 1191-6. doi:10.1016/0006-291X(02)00349-2. PMID    12054501.

In some embodiments, the Differentiation Inhibitor is chosen to havesolubility properties relative to the Stemness Driver that favors fasterrelease of the Differentiation Inhibitor relative to the Stemness Driverin an aqueous environment. In some embodiments, the DifferentiationInhibitor has a solubility in phosphate buffered saline that is 5, 10,50, 100, 500, 1000, or 5000-fold higher than that of the StemnessDriver.

TGF-β Inhibitors

In certain embodiments, the one or more additional agents comprises aTGFβ type I receptor inhibitor. Exemplary TGF-β Inhibitors appear inTable 5. TGF-beta type I receptor inhibitors include but are not limitedto 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole,which can be purchased from Calbiochem (San Diego). Other small moleculeinhibitors include, but are not limited to, SB-431542 (see e.g., Halderet al., 2005; Neoplasia 7(5):509-521), SM16 (see e.g., Fu, K et al.,2008; Arteriosclerosis, Thrombosis and Vascular Biology 28(4):665), andSB-505124 (see e.g., Dacosta Byfield, S., et al., 2004; MolecularPharmacology 65:744-52), among others.

TABLE 5 TGF-β Inhibitors Class Agent Alternative Name Tgf-beta-R1inhibitor LY-364947 616451, TGF-β RI Kinase Inhibitor I, CAS 396129-53-6, [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, ALK5 Inhibitor I,LY-364947, HTS-466284 Tgf-beta-R1 inhibitor Repsox 616452, TGF-β RIKinase Inhibitor II, CAS 446859- 33-2,2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)- 1,5-naphthyridineTgf-beta-R1 inhibitor SB-505124 616453, TGF-β RI Kinase Inhibitor III,CAS 356559- 13-22-(5-Benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl, ALK5 Inhibitor III, SB-505124, HClTgf-beta-R1 inhibitor A-83-01 616454, TGF-β RI Kinase Inhibitor IV - CAS909910- 43-6, 3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole, A-83-01, ALK5 Inhibitor IV Tgf-beta-R1inhibitor SD-208 616456, TGF-β RI Kinase Inhibitor V, CAS 627536- 09-8,2-(5-Chloro-2-fluorophenyl)pteridin-4- yl)pyridin-4-yl amine, SD-208,ALK5 Inhibitor V Tgf-beta-R1 inhibitor SB-431542 616461, TGF-β RI KinaseInhibitor VI, SB431542 - CAS 301836-41-9, 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol- 2-yl]benzamide,Dihydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate Tgf-beta-R1inhibitor TGF-β RI Kinase 616458, TGF-β RI Kinase Inhibitor VII - CASInhibitor VII 666729-57-3, 1-(2-((6,7-Dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone, ALK5 Inhibitor VIITgf-beta-R1 inhibitor SB-525334 616459, TGF-β RI Kinase Inhibitor VIII -CAS 356559-20-1, SB-525334, 6-(2-tert-Butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)- quinoxaline, ALK5 Inhibitor VIIITgf-beta-R1 inhibitor TGF-β RI Kinase 616463, TGF-β RI Kinase InhibitorIX, 4-((4-((2,6- Inhibitor IX Dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide, ALK5 Inhibitor IX Tgf-beta-R1 inhibitorGW788388 4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide Tgf-beta-R1 inhibitor LY21097617-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline Tgf-beta-R1 inhibitorGalunisertib 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- (LY2157299)pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6- carboxamide Tgf-beta-R1inhibitor EW-7197 N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2- methanamine Tgfbproduction Pirfenidone 2(1H)-Pyridinone, 5-methyl-1-phenyl- inhibitorTgf-beta-R1 inhibitor K02288 3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol Tgf-beta-R1 inhibitor D 44764-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide Tgf-beta-R1 inhibitor R 2687124-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol Other ITD 14-[1,1′-Biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester Smad3 inhibitorSIS3 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-[(2E)-3-(1-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1-oxo-2- propenyl]-isoquinolinehydrochloride Tgf-beta-R1 inhibitor A77-014-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4- yl]quinoline OtherAsiaticoside Tgf-beta-R1 inhibitor SM164-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1- carboxamide Tgf-betaantibody ID11 Tgf-beta antibody 2G7 Tgf-beta antibody GC-1008Fresolimumab Tgf-beta antibody CAT-152 Lerdelimimab Tgf-beta antibodyCAT-192 Metelimumab TGf-beta Receptor PF-03446962 antibody Tgf-betaantibody SR-2F Tgf-beta antibody 2G7 Tgf-beta antibody LY2382770Tgf-beta antibody IMC-TR1 Tgf-beta antibody STX-100 TGF-beta antagonistTGF-PRII: Fc Recombinant protein betaglycan/TGF- PRIII OligonucleotideAP12009 Trabedersen, antisense molecule inhibitor OligonucleotideAP11014 inhibitor Oligonucleotide AP15012 inhibitor LY-550410 LY-580276LY-364947 LY-2109761 LY-2157299 Galunisertib LY-573636 Is this TGF binhibitor/YES SB- 505124 SB-431542 SB-525234 SD-208 SD-093 Ki-26894NPC-30345 SX-007 IN-1130 pyrrole-imidazole Gene siliencing polyamideEW-7203 EW-7195 Structure EW-7197 GW6604 U.S. Pat. No. Pyrrolederivatives as pharmaceutical agents 7,087,626 U.S. Pat. No. Quinazolinederivatives as medicaments 6,476,031 U.S. Pat. No. Antibodies to TGF-β7,723,486, and EP 0945464 Peptide Trx-xFoxHIb Smad-interacting peptideaptamers Peptide Trx-Lefl Peptide Distertide (pI44) Peptide pI7 PeptideLSKL dihydropyrrlipyrazole- See U.S. Patent U.S. based scaffold Pat. No.8,298,825 B1 imidazole-based See U.S. Patent U.S. scaffold Pat. No.8,298,825 B1 pyrazolopyridine-based See U.S. Patent U.S. scaffold Pat.No. 8,298,825 B1 pyrazole-based scaffold See U.S. Patent U.S. Pat. No.8,298,825 B1 imidazopyridine-based See U.S. Patent U.S. scaffold Pat.No. 8,298,825 B1 triazole-based scaffold See U.S. Patent U.S. Pat. No.8,298,825 B1 pyridopyrimidine-based See U.S. Patent U.S. scaffold Pat.No. 8,298,825 B1 pyrrolopyrazole-based See U.S. Patent U.S. scaffoldPat. No. 8,298,825 B1 isothiazole-based See U.S. Patent U.S. scaffoldPat. No. 8,298,825 B1 oxazole-based scaffold See U.S. Patent U.S. Pat.No. 8,298,825 B1 Tgf-beta-R1 inhibitor Repsox 616452, TGF-β RI KinaseInhibitor II, CAS 446859- 33-2,2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)- 1,5-naphthyridineTgf-beta-R1 inhibitor Galunisertib4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- (LY2157299)pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6- carboxamide Tgf-beta-R1inhibitor EW-7197 N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2- methanamine Tgfbproduction Pirfenidone 2(1H)-Pyridinone, 5-methyl-1-phenyl- inhibitorLY-2157299 Galunisertib Tgf-beta-R1 inhibitor SB-505124 616453, TGF-β RIKinase Inhibitor III, CAS 356559-13-22-(5-Benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl, ALK5 Inhibitor III, SB-505124, HClTgf-beta-R1 inhibitor SB-525334 616459, TGF-β RI Kinase Inhibitor VIII -CAS 356559-20-1, SB-525334, 6-(2-tert-Butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)- quinoxaline, ALK5 Inhibitor VIIITgf-beta-R1 inhibitor TGF-β RI Kinase 616463, TGF-β RI Kinase InhibitorIX, 4-((4-((2,6- Inhibitor IX Dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide, ALK5 Inhibitor IX Tgf-beta-R1 inhibitor R268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol SB- 505124 Pyridine,2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-, hydrochloride (1:1) SD-208IN-1130 EW-7197 Tgf-beta-R1 inhibitor A-83-01 616454, TGF-β RI KinaseInhibitor IV - CAS 909910- 43-6,3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole, A-83-01, ALK5 Inhibitor IV Tgf-beta-R1inhibitor SB-431542 616461, TGF-β RI Kinase Inhibitor VI, SB431542 - CAS301836-41-9, 4-[4-(3,4- Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate Tgf-beta-R1inhibitor R 268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol

Additional Therapeutic Agents

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population one or moreadditional agents (e.g., in addition to (i) and (ii)). In certainembodiments, the one or more additional agents comprises an ROSinhibitor or scavenger. ROS scavengers include but are not limited toenzymes catalase, glutathione peroxidase and ascorbate peroxidase.Additionally, vitamins A, E, and C are known to have scavenger activity.Minerals such as selenium and manganese can also be efficaciouscompounds for scavenging ROS.

ROS inhibitors include but are not limited to alpha lipoic acid, asuperoxide dismutase mimetic, or a catalase mimetic. The superoxidedismutase mimetic or the catalase mimetic can be MnTBAP(Mn(III)tetrakis(4-benzoic acid)porphyrin chloride)(produced byCalbiochem), ZnTBAP (Zn(III)tetrakis(4-benzoic acid)porphyrin chloride),SC-55858 (manganese (11) dichloro(2R,3R,8R,9R-bis-cyclohexano-1,4,7,10,13-pentaazacyclopentadecane)]Euk-134 (3,3′-methoxysalenMn(III)) (produced by Eukarion), M40403(dichloro [(4aR,13 aR,17aR,21 aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21-eicosahydro-11,7-nitrilo-7H-dibenzo[1,4,7,10]tetraazacycloheptadecine-kappaNS,kappaN13,kappaN18,kappaN21,kappaN22]manganese) (produced by Metaphore), AEOL 10112, AEOL 10113, and AEOL10150 (manganese(III) mesotetrakis (di-N-ethylimidazole) porphyrin)(allAEOL compounds being produced by Incara Pharmaceuticals). Alternatively,the ROS inhibitor can be an iron chelator. Of the iron chelators,deferoxamine or DFO may be the most important, because it isFDA-approved for treatment of iron excess in thallasemia. In addition,the ROS inhibitor can be a composition comprised of a mixture of ironchelators.

ROS inhibitors may be radiation protectors, and include such compoundsas, for example, uric acid, buthionine sulfoximine, diethyl maleate,vitamin E, vitamin C, cysteine such as N-acetyl cysteine, orglutathione, metronidazole, and a retinoid such as, e.g., vitamin A.Additional ROS scavengers may be found at

In certain embodiments, the one or more additional agents comprisesvitamin C or a derivative thereof. In certain embodiments, the one ormore additional agents comprises a TGFβ type I receptor inhibitor.TGF-beta type I receptor inhibitors include but are not limited to2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole,which can be purchased from Calbiochem (San Diego, Calif.). Other smallmolecule inhibitors include, but are not limited to, SB-431542 (seee.g., Halder et al., 2005; Neoplasia 7(5):509-521), SM16 (see e.g., Fu,K et al., 2008; Arteriosclerosis, Thrombosis and Vascular Biology28(4):665), and SB-505124 (see e.g., Dacosta Byfield, S., et al., 2004;Molecular Pharmacology 65:744-52), among others.

In one embodiment, the ALK5 inhibitor2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine is usedwith the methods described herein. This inhibitor is also referred toherein as ALK5 inhibitor II and is available commercially fromCalbiochem (Cat. No. 616452; San Diego, Calif.). In one embodiment, theinhibitor is SB 431542, an ALK-4, -5, -7inhibitor, commerciallyavailable from Sigma (product no. 54317; Saint Louis, Mo.). SB 431542 isalso referred to by the following chemical names:4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide,4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide,or4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamidehydrate.

Small molecules inhibitors of TGF-β signaling can be classified based onthe basic scaffold of the molecule. For example, TGF-β signalinginhibitors can be based on the dihydropyrrlipyrazole-based scaffold,imidazole-based scaffold, pyrazolopyridine-based scaffold,pyrazole-based scaffold, imidazopyridine-based scaffold, triazole-basedscaffold, pyridopyrimidine-based scaffold, pyrrolopyrazole-basedscaffold, isothiazole-based scaffold and oxazole-based scaffold.

Inhibitors of TGF-β signaling are described, for example, in Callahan,J. F. et al., J. Med. Chem. 45, 999-1001 (2002); Sawyer, J. S. et al.,J. Med. Chem. 46, 3953-3956 (20031; Gellibert, F. et al., J. Med. Chem.47, 4494-4506 (2004); Tojo, M. et al., Cancer Sci. 96: 791-800 (2005);Valdimarsdottir, G. et al., APMIS 113, 773-389 (2005); Petersen et al.Kidney International 73, 705-715 (2008); Yingling, J. M. et al., NatureRev. Drug Disc. 3, 1011-1022 (2004); Byfield, S. D. et al., Mol.Pharmacol., 65, 744-752 (2004); Dumont, N, et al., Cancer Cell 3,531-536 (2003); WO Publication No. 2002/094833; WO Publication No.2004/026865; WO Publication No. 2004/067530; WO Publication No.209/032667; WO Publication No. 2004/013135; WO Publication No.2003/097639; WO Publication No. 2007/048857; WO Publication No.2007/018818; WO Publication No. 2006/018967; WO Publication No.2005/039570; WO Publication No. 2000/031135; WO Publication No.1999/058128; U.S. Pat. Nos. 6,509,318; 6,090,383; 6,419,928; 9,927,738;7,223,766; 6,476,031; 6,419,928; 7,030,125; 6,943,191; U.S. PublicationNo. 2005/0245520; U.S. Publication No. 2004/0147574; U.S. PublicationNo. 2007/0066632; U.S. Publication No. 2003/0028905; U.S. PublicationNo. 2005/0032835; U.S. Publication No. 2008/0108656; U.S. PublicationNo. 2004/015781; U.S. Publication No. 2004/0204431; U.S. Publication No.2006/0003929; U.S. Publication No. 2007/0155722; U.S. Publication No.2004/0138188 and U.S. Publication No. 2009/0036382, the contents of eachwhich are herein incorporated by reference in their entirety.

Oligonucleotide based modulators of TGF-β signaling, such as siRNAs andantisense oligonucleotides, are described in U.S. Pat. Nos. 5,731,424;6,124,449; U.S. Publication Nos. 2008/0015161; 2006/0229266;2004/0006030; 2005/0227936 and 2005/0287128, each of which are hereinincorporated by reference in their entirety. Other antisense nucleicacids and siRNAs can be obtained by methods known to one of ordinaryskill in the art.

Exemplary inhibitors of TGF-β signaling include, but are not limited to,AP-12009 (TGF-β Receptor type II antisense oligonucleotide),Lerdelimumab (CAT 152, antibody against TGF-β Receptor type II) GC-1008(antibody to all isoforms of human TGF-β), ID11 (antibody to allisoforms of murine TGF-0), soluble TGF-β, soluble TGF-β Receptor typeII, dihydropyrroloimidazole analogs (e.g., SKF-104365), triarylimidazoleanalogs (e.g., SB-202620(4-(4-(4-fluorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)benzoic acid)and SB-203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole)), RL-0061425, 1,5-naphthyridineaminothiazole and pyrazole derivatives (e.g.,4-(6-methyl-pyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazole-2-amineand 2-[3-(6-methyl-pyridin-2-yl)-1H-pyrazole-4-yl]-1,5-naphthyridine),SB-431542(4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide),GW788388(4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide),A-83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide),Decorin, Lefty 1, Lefty 2, Follistatin, Noggin, Chordin, Cerberus,Gremlin, Inhibin, BIO (6-bromo-indirubin-3′-oxime), Smad proteins (e.g.,Smad6, Smad7), and Cystatin C.

Inhibitors of TGF-β signaling also include molecules which inhibit TGF-βReceptor type I. Inhibitors of TGF-β Receptortype I are described inByfield, S. D., and Roberts, A. B., Trends Cell Biol. 14, 107-111(2004); Sawyer J. S. et al., Bioorg. Med. Chem. Lett. 14, 3581-3584(2004); Sawyer, J. S. et al., J. Med. Chem. 46, 3953-3956 (2003);Byfield, S. D. et al., Mol. Pharmacol. 65, 744-752 (2004); Gellibert, F.et al., J. Med. Chem. 47, 4494-4506 (2004); Yingling, J. M. et al.,Nature Rev. Drug Disc. 3, 1011-1022 (2004); Dumont, N, et al., CancerCell 3, 531-536 (2003); Tojo, M. et al., Cancer Sci. 96: 791-800 (2005);WO Publication No. 2004/026871; WO Publication No. 2004/021989; WOPublication No. 2004/026307; WO Publication No. 2000/012497; U.S. Pat.Nos. 5,731,424; 5,731,144; 7,151,169; U.S. Publication No. 2004/00038856and U.S. Publication No. 2005/0245508, contents of all of which areherein incorporated in their entireties.

Combinations of Agents

In certain embodiments, the composition comprises an agent within theclasses of Table 1, Column A (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the classes of Table 3,Column A (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theclasses of Table 1, Column A (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the classes of Table 4,Column A (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theclasses of Table 2, Column A (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the classes of Table 3,Column A (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theclasses of Table 2, Column A (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the classes of Table 4,Column A (or a derivative or pharmaceutically acceptable salt thereof).

In certain embodiments, the composition comprises an agent within theagents of Table 1, Column B (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the agents of Table 3,Column B (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theagents of Table 1, Column B (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the agents of Table 4,Column B (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theagents of Table 2, Column B (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the agents of Table 3,Column B (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises an agent within theagents of Table 2, Column B (or a derivative or pharmaceuticallyacceptable salt thereof) and an agent within the agents of Table 4,Column B (or a derivative or pharmaceutically acceptable salt thereof).

In certain embodiments, the composition comprises a combination ofagents including (i) a GSK3-beta inhibitor and/or Wnt agonist (or aderivative or pharmaceutically acceptable salt thereof), and (ii) anotch agonist and/or HDAC inhibitor, where these agents are drawn fromTable 1-4, respectively (or a derivative or pharmaceutically acceptablesalt thereof).

In certain embodiments, the composition comprises (i) a GSK3-betainhibitors drawn from aminopyrimidines, inorganic atoms, orthiadiazolidindiones (or a derivative or pharmaceutically acceptablesalt thereof) and (ii) a notch agonist and/or HDAC inhibitor drawn fromTable 3-4 (or a derivative or pharmaceutically acceptable salt thereof).In certain embodiments, the composition comprises (i) a Wnt agonistdrawn from GSK3-beta inhibitors, Wnt ligand, or Wnt related protein (ora derivative or pharmaceutically acceptable salt thereof) and (ii) anotch agonist and/or HDAC inhibitor drawn from Table 3-4 (or aderivative or pharmaceutically acceptable salt thereof). In certainembodiments, the composition comprises (i) a Notch agonist drawn fromHDAC inhibitors, or Natural receptor ligand (or a derivative orpharmaceutically acceptable salt thereof) and (ii) a GSK3-beta inhibitorand/or Wnt agonist drawn from Table 1-2 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises an HDAC inhibitor drawn from Hydroxamates,Aliphatic Acid, or Benzamides (or a derivative or pharmaceuticallyacceptable salt thereof) and (ii) a GSK3-beta inhibitor and/or Wntagonist drawn from Table 1-2 (or a derivative or pharmaceuticallyacceptable salt thereof). In certain embodiments, the compositioncomprises a combination of agents including (i) a GSK3-beta inhibitorand/or Wnt agonist (or a derivative or pharmaceutically acceptable saltthereof) and (ii) a notch agonist and/or HDAC inhibitor (or a derivativeor pharmaceutically acceptable salt thereof), where these agents aredrawn from Table 1-4, respectively.

In certain embodiments, the composition comprises (i) a GSK3-betainhibitors drawn from CHIR99021, Lithium, or NP031112(Tideglusib) (or aderivative or pharmaceutically acceptable salt thereof) and (ii) a notchagonist and/or HDAC inhibitor drawn from Table 3-4 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises (i) a Wnt agonist drawn from CHIR99021, Wnt3a, orR-spondin1 (or a derivative or pharmaceutically acceptable salt thereof)and (ii) a notch agonist and/or HDAC inhibitor drawn from Table 3-4 (ora derivative or pharmaceutically acceptable salt thereof). In certainembodiments, the composition comprises (i) a Notch agonist drawn fromValproic Acid, SAHA (vorinostat), Jagged 1, Delta-like1, or Delta-like 4(or a derivative or pharmaceutically acceptable salt thereof) and (ii) aGSK3-beta inhibitor and/or Wnt agonist drawn from Table 1-2 (or aderivative or pharmaceutically acceptable salt thereof). In certainembodiments, the composition comprises an HDAC inhibitor drawn fromValproic Acid, SAHA (vorinostat), or Tubastatin A (or a derivative orpharmaceutically acceptable salt thereof) and (ii) a GSK3-beta inhibitorand/or Wnt agonist drawn from Table 1-2 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises a combination of agents including (i) a GSK3-betainhibitor and/or Wnt agonist (or a derivative or pharmaceuticallyacceptable salt thereof) and (ii) a notch agonist and/or HDAC inhibitor,where these agents are drawn from Table 1-4 (or a derivative orpharmaceutically acceptable salt thereof), respectively.

In certain embodiments, the composition comprises (i) a GSK3-betainhibitors drawn from aminopyrimidines, inorganic atoms, orthiadiazolidindiones (or a derivative or pharmaceutically acceptablesalt thereof) and (ii) a highly potent notch agonist and/or highlypotent HDAC inhibitor drawn from Table 3-4 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises (i) a Wnt agonist drawn from GSK3-beta inhibitors,Wnt ligand, or Wnt related protein (or a derivative or pharmaceuticallyacceptable salt thereof) and (ii) a highly potent notch agonist and/orhighly potent HDAC inhibitor drawn from Table 3-4 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises (i) a Notch agonist drawn from HDAC inhibitors, orNatural receptor ligand (or a derivative or pharmaceutically acceptablesalt thereof) and (ii) a highly potent GSK3-beta inhibitor and/or highlypotent Wnt agonist drawn from Table 1-2 (or a derivative orpharmaceutically acceptable salt thereof). In certain embodiments, thecomposition comprises an HDAC inhibitor drawn from Hydroxamates,Aliphatic Acid, or Benzamides (or a derivative or pharmaceuticallyacceptable salt thereof) and (ii) a highly potent GSK3-beta inhibitorand/or highly potent Wnt agonist drawn from Table 1-2 (or a derivativeor pharmaceutically acceptable salt thereof).

In certain embodiments, the composition comprises (i) a GSK3-betainhibitors drawn from CHIR99021, Lithium, or NP031112(Tideglusib) (or aderivative or pharmaceutically acceptable salt thereof) and (ii) ahighly potent notch agonist and/or highly potent HDAC inhibitor drawnfrom Table 3-4 (or a derivative or pharmaceutically acceptable saltthereof). In certain embodiments, the composition comprises (i) a Wntagonist drawn from CHIR99021, Wnt3a, or R-spondin1 (or a derivative orpharmaceutically acceptable salt thereof) and (ii) a highly potent notchagonist and/or highly potent HDAC inhibitor drawn from Table 3-4 (or aderivative or pharmaceutically acceptable salt thereof). In certainembodiments, the composition comprises (i) a Notch agonist drawn fromValproic Acid, SAHA (vorinostat), Jagged 1, Delta-like1, or Delta-like 4(or a derivative or pharmaceutically acceptable salt thereof) and (ii) ahighly potent GSK3-beta inhibitor and/or highly potent Wnt agonist drawnfrom Table 1-2 (or a derivative or pharmaceutically acceptable saltthereof). In certain embodiments, the composition comprises an HDACinhibitor drawn from Valproic Acid, SAHA (vorinostat), or Tubastatin A(or a derivative or pharmaceutically acceptable salt thereof) and (ii) ahighly potent GSK3-beta inhibitor and/or highly potent Wnt agonist drawnfrom Table 1-2 (or a derivative or pharmaceutically acceptable saltthereof).

In certain embodiments, the composition comprises a combination ofagents including (i) a GSK3-beta inhibitor and/or Wnt agonist (or aderivative or pharmaceutically acceptable salt thereof), and a secondagent which is unique from the first agent and is (ii) a notch agonistand/or HDAC inhibitor (or a derivative or pharmaceutically acceptablesalt thereof).

In certain embodiments, the composition comprises a combination ofagents including (i) a highly potent GSK3-beta inhibitor and/or highlypotent Wnt agonist (or a derivative or pharmaceutically acceptable saltthereof) and (ii) a highly potent notch agonist and/or highly potentHDAC inhibitor (or a derivative or pharmaceutically acceptable saltthereof). In certain embodiments the composition comprises a combinationof agents including (i) a highly potent GSK3-beta inhibitor and/orhighly potent Wnt agonist (or a derivative or pharmaceuticallyacceptable salt thereof) and (ii) a notch agonist and/or HDAC inhibitor(or a derivative or pharmaceutically acceptable salt thereof). Incertain embodiments, the composition comprises a combination of agentsincluding (i) a GSK3-beta inhibitor and/or Wnt agonist (or a derivativeor pharmaceutically acceptable salt thereof), and (ii) a highly potentnotch agonist and/or highly potent HDAC inhibitor (or a derivative orpharmaceutically acceptable salt thereof).

In certain embodiments, the composition comprises a Stemness Driver at5, 10, 20, 50, 100, 200, 500, 1000, or 5000 times the Effective StemnessDriver Concentration. In some embodiments, the composition furthercomprises the Differentiation Inhibitor at 5, 10, 20, 50, 100, 200, 500,1000, or 5000 times the Effective Differentiation InhibitionConcentration. Optionally, any of the previous composition may includeone or more agents that target the ROS or TgfBeta. Optionally, any ofthe previous composition may include one or more neurotrophins.

Delivery Profile of Combined Agents

In some embodiments, a Stemness Driver may be used to drive theproliferation of Lgr5′ stem cells. In some cases, a Stemness Driver mayalso induce differentiation of LGR5+ cells to hair cells if aDifferentiation Inhibitor is not present at an Effective DifferentiationInhibition Concentration. Examples of Stemness Drivers that may driveboth proliferation and differentiation include GSK3Beta inhibitors andWnt agonists. In some embodiments, the Differentiation Inhibitor may bea Notch agonist or HDAC inhibitor. In some embodiments, there may be afirst Proliferation Period with an Effective Stemness DriverConcentration and an Effective Differentiation Inhibition Concentrationof a Differentiation Inhibitor, followed by a Differentiation Periodwith an Effective Stemness Driver Concentration and without an EffectiveDifferentiation Inhibition Concentration of a Differentiation Inhibitor.In some embodiments, there may be a first Proliferation Period with anEffective Stemness Driver Concentration of a Wnt agonist or GSK3Betainhibitor and an Effective Differentiation Inhibition Concentration of aNotch agonist or HDAC inhibitor, followed by a Differentiation Periodwith an Effective Sternness Driver Concentration of a Wnt agonist orGSK3Beta inhibitor and without an Effective Differentiation InhibitionConcentration of a Notch agonist or HDAC inhibitor. In some embodiments,there may be a first Proliferation Period with an Effective StemnessDriver Concentration of a GSK3Beta inhibitor and an EffectiveDifferentiation Inhibition Concentration of an HDAC inhibitor, followedby a Differentiation Period with an Effective Stemness DriverConcentration of a GSK3Beta inhibitor and without an EffectiveDifferentiation Inhibition Concentration of an HDAC inhibitor.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a compositionmaintains an Effective Release Rate of Stemness Driver throughout thedesired Proliferation Period. In some embodiments, a compositionmaintains an Effective Release Rate of Stemness Driver for at least 1hour. In some embodiments, it is desired to have a Stemness Driverrelease rate of 10, 20, 50, 100, 500, or 1000-fold the Effective ReleaseRate of Stemness Driver for the desired proliferation period.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition placedon the round window membrane of a mouse retains an Effective StemnessDriver Concentration in the cochlea throughout the desired ProliferationPeriod. In some embodiments, a composition placed on the round windowmembrane of a mouse retains an Effective Stemness Driver Concentrationin the cochlea for at least 1 hour. In some embodiments, a compositionplaced on the round window membrane of a mouse retains an EffectiveStemness Driver Concentration for at least 2, 4, 8, 16, 24, 48, 72, 96,or 192 hours.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a compositionmaintains an Effective Release Rate of Differentiation Inhibitorthroughout the desired Proliferation Period. In some embodiments, acomposition maintains an Effective Release Rate of DifferentiationInhibitor for at least 1 hour. In some embodiments, a composition placedon the round window membrane of a mouse retains an Effective ReleaseRate of Differentiation Inhibitor for at least 2, 4, 8, 16, 24, 48, 72,96, or 192 hours.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition placedon the round window membrane of a mouse retains an EffectiveDifferentiation Inhibition Concentration of a Differentiation Inhibitorin the cochlea throughout the desired Proliferation Period. In someembodiments, a composition placed on the round window membrane of amouse retains an Effective Differentiation Inhibition Concentration inthe cochlea for at least 1 hour. In some embodiments, a compositionplaced on the round window membrane of a mouse retains an EffectiveDifferentiation Inhibition Concentration for at least 2, 4, 8, 16, 24,48, 72, 96, or 192 hours.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition mayrelease both a Stemness Driver and a Differentiation Inhibitorsimultaneously. It may be advantageous to have the DifferentiationInhibitor mitigate the degree to which the Stemness Driver reduces Notchactivity in the Lgr5+ cells targets for proliferation by the therapy. Insome embodiments, the composition has a Release Rate of Stemness Driverand Differentiation Inhibitor throughout the Proliferation Period thatif the mass of agent release in 1 hour is placed in 30 ul, and added toa Notch Activity Assay in cell culture, the Notch Activity would beat >20, 30, 40, 50, 60, 70, 80, or 90 of the Notch Activity of nativestate without the agents being applied.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition mayrelease both a Stemness Driver and a Differentiation Inhibitorsimultaneously. In some embodiments, a desired composition place on theround window membrane of a mouse releases an amount of a Stemness Driverand a Differentiation Inhibitor simultaneously to maintain NotchActivity at >20, 30, 40, 50, 60, 70, 80, or 90 of the Notch Activity ofnative state without the agents being applied.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition mayrelease both a Stemness Driver and a Differentiation Inhibitorsimultaneously. It may be advantageous to have the DifferentiationInhibitor mitigate the degree to which the Stemness Driver reduces Notchactivity in the Lgr5+ cells targets for proliferation by the therapy. Insome embodiments, the composition has a Release Rate of Stemness Driverand Differentiation Inhibitor throughout the Proliferation Period thatif the mass of agent release in 1 hour is placed in 30 ul, and added toa Notch Activity Assay in cell culture, the Notch Activity is >2, 3, 4,5, 10, 20, 50, 100, 500, 100× of the Notch Activity as that which wouldhave achieved if the composition had contained the same amount ofStemness Driver without any Differentiation Inhibitor.

In some embodiments, the desired Proliferation period is 1, 2, 4, 8, 16,24, 48, 72, 96, or 192 hours. In some embodiments, a composition mayrelease both a Stemness Driver and a Differentiation Inhibitorsimultaneously. It may be advantageous to have the DifferentiationInhibitor mitigate the degree to which the Stemness Driver reduces Notchactivity in the Lgr5⁺ cells targets for proliferation by the therapy. Insome embodiments, when a composition comprising Stemness Driver and aDifferentiation Inhibitor are placed on the round window membrane of amouse, the Notch activity of Lgr5⁺ cells in the cochlea are >2, 3, 4, 5,10, 20, 50, 100, 500, 100× of the Notch activity as that which wouldhave achieved if the composition had contained the same amount ofStemness Driver without any Differentiation Inhibitor.

In some embodiments, the desired Differentiation period is 1, 2, 4, 8,16, 24, 48, 72, 96 days. In some embodiments, a composition does notachieve an Effective Release Rate of Differentiation Inhibitor at anytime in the desired Differentiation Period. In some embodiments, acomposition does not achieve an Effective Release Rate ofDifferentiation Inhibitor for more than 1 day. In some embodiments, acomposition does not achieve an Effective Release Rate ofDifferentiation Inhibitor for at least 2, 4, 8, 16, 24, 48, 72, or 96days.

In some embodiments, the desired Differentiation period is 1, 2, 4, 8,16, 24, 48, 72, 96 days. In some embodiments, a composition placed onthe round window membrane of a mouse does not retain an EffectiveDifferentiation Inhibition Concentration of a Differentiation Inhibitorat any time in the desired Differentiation Period. In some embodiments,a composition placed on the round window membrane of a mouse does notretain an Effective Differentiation Inhibition Concentration for morethan 1 day. In some embodiments, a composition does not retain anEffective Differentiation Inhibition Concentration for at least 2, 4, 8,16, 24, 48, 72, or 96 days.

In some embodiments, it is desirable to release the Stemness Driver overa longer period of time than the Differentiation inhibitor. In someembodiments, the Mean Release Time of Stemness Driver is 2, 4, 8, 16, or32 times great than the Mean Release Time of the DifferentiationInhibitor.

In certain embodiments, the stem cell population comprises supportingcells. In certain embodiments, the supporting cells are Lgr5⁺ cells. Incertain embodiments, the stem cell population comprises post-natalcells. In certain embodiments, the hair cells are inner ear hair cells.In certain embodiments, the hair cells are outer ear hair cells.

In certain embodiments, stem cells include progenitor cells.

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population a notchagonist that is also an HDAC inhibitor. In certain embodiments, theadministering step comprises administering or causing to be administeredto the stem cell population a notch agonist that comprises a syntheticmolecule. In certain embodiments, the administering step comprisesadministering or causing to be administered to the stem cell populationvalproic acid (VPA) (e.g., in a pharmaceutically acceptable form (e.g.,salt)) (e.g., where VPA is a notch agonist that is also an HDACinhibitor).

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population a Wnt agonistthat is also a GSK3-beta inhibitor. In certain embodiments, theadministering step comprises administering or causing to be administeredto the stem cell population a Wnt agonist that comprises a syntheticmolecule. In certain embodiments, the administering step comprisesadministering or causing to be administered to the stem cell populationCHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., salt))(e.g., where CHIR99021 is a GSK3-beta inhibitor.

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population a notchinhibitor. In certain embodiments, the notch inhibitor comprises DAPT,LY411575, MDL-28170, Compound E, R04929097; DAPT(N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester); L-685458((5S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L-leu-L-phe-amide);BMS-708163 (Avagacestat); BMS-299897(2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoicacid); M-0752; YO-01027; MDL28170 (Sigma); LY41 1575(N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yI)-1-alaninamide);ELN-46719 (2-hydroxy-valeric acid amide analog of LY41 1575; PF-03084014((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide);Compound E((2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide;and Semagacestat (LY450139; (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-2-oxoethyl)butanamide),or pharmaceutically acceptable salts thereof. (e.g., in apharmaceutically acceptable form (e.g., salt)),

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population: (i) CHIR99021(e.g., in a pharmaceutically acceptable form (e.g., salt)) and (ii) VPA(e.g., in a pharmaceutically acceptable form (e.g., salt)) (e.g., where(i) comprises CHIR99021 and (ii) comprises VPA). In certain embodiments,the administering step further comprises administering or causing to beadministered to the stem cell population DAPT (e.g., where DAPT is anotch inhibitor).

In certain embodiments, the administering step is carried out byperforming one or more injections into the ear (e.g.,transtympanically). In certain embodiments, the one or more injectionsare into the middle ear. In certain embodiments, the one or moreinjections are into the inner ear. In certain embodiments, performingthe one or more injections comprises anesthetizing the tympanic membraneand/or surrounding tissue, placing a needle through the tympanicmembrane into the middle ear, and injecting one or both of (i) and (ii).

In certain embodiments, the administering step comprises administeringthe notch agonist and/or HDAC inhibitor in a pulsatile manner andadministering the GSK3-beta inhibitor and/or Wnt agonist in a sustainedmanner. In certain embodiments, the formulation administered is sterile.In certain embodiments, the formulation administered is pyrogen-free. Incertain embodiments, a pharmaceutically acceptable formulation isadministered as described in Appendix pages 21-36. In certainembodiments, the formulation is a combination of (i) a GSK3-betainhibitor and/or Wnt agonist, and (ii) a notch agonist and/or HDACinhibitor administered in a pharmaceutically acceptable formulation asdescribed in Appendix pages 21-36.

In certain embodiments, the stem cell population is of an in vivosubject, and the method is a treatment for hearing loss and/orvestibular dysfunction (e.g., wherein the generation of inner ear haircells from the expanded population of stem cells results in partial orfull recovery of hearing loss and/or improved vestibular function). Incertain embodiments, the stem cell population is of an in vivo subject,and the method further comprises delivering a drug to the subject (e.g.,for treatment of a disease and/or disorder unrelated to hearing lossand/or vestibular dysfunction) at a higher concentration than a knownsafe maximum dosage of the drug for the subject (e.g., the known safemaximum dosage if delivered in the absence of the generation of innerear hair cells resulting from the method) (e.g., due to a reduction orelimination of a dose-limiting ototoxicity of the drug).

In certain embodiments, the method further comprises performing highthroughput screening using the generated inner ear hair cells. Incertain embodiments, the method comprises using the generated inner earhair cells to screen molecules for toxicity against inner ear haircells. In certain embodiments, the method comprises using the generatedinner ear hair cells to screen molecules for ability to improve survivalof inner ear hair cells (e.g., inner ear hair cells exposed to saidmolecules).

In another aspect, the disclosure is directed to a method of producingan expanded population of stem cells, the method comprising:administering or causing to be administered to a stem cell population(e.g., of an in vitro, ex vivo, or in vivo sample/subject) both of (i)and (ii): (i) a GSK3-beta inhibitor and/or Wnt agonist, and (ii) a notchagonist and/or HDAC inhibitor, thereby proliferating stem cells in thestem cell population and resulting in an expanded population of stemcells. In certain embodiments, the stem cell population comprises Lgr5+cells. In certain embodiments, the stem cell population comprisespost-natal stem cells. In certain embodiments, the stem cell populationcomprises epithelial stem cells. In certain embodiments, stem cellsinclude progenitor cells.

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population a notchagonist that is also an HDAC inhibitor. In certain embodiments, theadministering step comprises administering or causing to be administeredto the stem cell population a notch agonist that comprises a syntheticmolecule. In certain embodiments, the administering step comprisesadministering or causing to be administered to the stem cell populationVPA (e.g., in a pharmaceutically acceptable form (e.g., salt)) (e.g.,where VPA is a notch agonist that is also an HDAC inhibitor).

In certain embodiments, the administering step comprises administeringor causing to be administered to the stem cell population a Wnt agonistthat is also a GSK3-beta inhibitor. In certain embodiments, theadministering step comprises administering or causing to be administeredto the stem cell population a Wnt agonist comprising a syntheticmolecule. In certain embodiments, the administering step comprisesadministering or causing to be administered to the stem cell populationCHIR99021 (e.g., in a pharmaceutically acceptable form (e.g., salt))(e.g., where CHIR99021 is a GSK3 beta inhibitor).

In certain embodiments, the administering step is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In certain embodiments, the administering step comprises administeringthe notch agonist and/or HDAC inhibitor in a pulsatile manner andadministering the GSK3-beta inhibitor and/or Wnt agonist in a sustainedmanner.

In certain embodiments, the stem cells are inner ear stem cells and/orsupporting cells.

In certain embodiments, the method further comprises performing highthroughput screening using the generated expanded population of stemcells. In certain embodiments, the method further comprises using thegenerated stem cells to screen molecules for toxicity against stem cellsand/or their progeny. In certain embodiments, the method comprises usingthe generated stem cells to screen molecules for ability to improvesurvival of stem cells and/or their progeny.

In another aspect, the disclosure is directed to a method of treating asubject who has, or is at risk of developing, hearing loss and/orvestibular dysfunction, the method comprising: identifying a subject whohas experienced, or is at risk for developing, hearing loss and/orvestibular dysfunction, administering or causing to be administered tothe subject both of (i) and (ii): (i) a GSK3-beta inhibitor and/or Wntagonist, and (ii) a notch agonist and/or HDAC inhibitor, therebytreating or preventing the hearing loss and/or vestibular dysfunction inthe subject.

In certain embodiments, the stem cell population comprises Lgr5⁺ cells.In certain embodiments, the stem cell population comprises post-natalcells. In certain embodiments, the stem cell population comprisesepithelial stem cells. In certain embodiments, stem cells includeprogenitor cells.

In certain embodiments, the administering step comprises administeringor causing to be administered to the subject a notch agonist that isalso an HDAC inhibitor. In certain embodiments, the administering stepcomprises administering or causing to be administered to the subject anotch agonist comprising a synthetic molecule. In certain embodiments,the administering step comprises administering or causing to beadministered to the subject VPA (e.g., in a pharmaceutically acceptableform (e.g., salt)) (e.g., where VPA is a notch agonist that is also anHDAC inhibitor).

In certain embodiments, the administering step comprises administeringor causing to be administered to the subject a Wnt agonist that is alsoa GSK3-beta inhibitor. In certain embodiments, the administering stepcomprises administering or causing to be administered to the subject aWnt agonist comprising a synthetic molecule. In certain embodiments, theadministering step comprises administering or causing to be administeredto the subject CHIR99021 (e.g., in a pharmaceutically acceptable form(e.g., salt)) (e.g., where CHIR99021 is a GSK3-beta inhibitor).

In certain embodiments, the step of administering is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In certain embodiments, the method comprises administering the notchagonist and/or the HDAC inhibitor in a pulsatile manner andadministering the GSK3-beta inhibitor and/or Wnt agonist in a sustainedmanner.

In another aspect, the disclosure is directed to a kit comprising: (a) aset of one or more compositions, the set comprising (i) and (ii): (i) aGSK3-beta inhibitor and/or Wnt agonist, and (ii) a notch agonist and/orHDAC inhibitor, each of the one or more compositions provided in apharmaceutically acceptable carrier and (b) instructions for using theset of one or more compositions to treat an inner ear disorder.

In certain embodiments, the set of one or more compositions alsocomprises a TGFβ inhibitor. In certain embodiments, the set of one ormore compositions also comprises an ROS scavenger. In certainembodiments, the ROS scavenger is vitamin C or a derivative thereof. Incertain embodiments, the set of one or more compositions is/are in aform that can be injected (e.g. via syringe). In certain embodiments,the set of one or more compositions is/are in a form that can beinjected into the middle ear.

In another aspect, the disclosure is directed to a pharmaceuticalcomposition comprising a GSK3-beta inhibitor and a notch agonist inlyophilized form.

In another aspect, the disclosure is directed to a pharmaceuticalcomposition comprising a GSK3-beta inhibitor and a notch agonist inhydrated form.

In certain embodiments, the GSK3-beta inhibitor is CHIR99021 (e.g., in apharmaceutically acceptable form (e.g., salt)). In certain embodiments,the notch agonist is VPA (e.g., in a pharmaceutically acceptable form(e.g., salt)).

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating stem cells inan initial stem cell population (e.g., of an in vitro, ex vivo, or invivo sample/subject), resulting in an expanded population of stem cells(e.g., such that the expanded population is a factor of at least 1.25,1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation); and exposing the expanded population of stem cells to aGSK3-beta inhibitor and/or a Wnt agonist, and, optionally, a notchinhibitor, thereby facilitating generation of inner ear hair cells fromthe expanded population of stem cells.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising administering CHIR99021(e.g., in a pharmaceutically acceptable form (e.g., salt)) to a cellpopulation in an inner ear of a subject, thereby facilitating generationof inner ear hair cells.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating post-natalLGR5+ cells in an initial population (e.g., of an in vitro, ex vivo, orin vivo sample/subject), resulting in an expanded population of LGR5+cells (e.g., such that the expanded population is a factor of at least1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation), said expanded population of LGR5+ cells resulting ingeneration of inner ear hair cells. In certain embodiments, stem cellsinclude progenitor cells.

In another aspect, the disclosure is directed to a method of treating adisease or disorder, the method comprising: proliferating post-natalLgr5⁺ epithelial cells in an initial population of a subject (in vivo),resulting in an expanded population of Lgr5+ epithelial cells (e.g.,such that the expanded population is a factor of at least 1.25, 1.5,1.75, 2, 3, 5, 10, or 20 greater than the initial post-natal Lgr5⁺epithelial cell population).

In some embodiments, Lgr5⁺ cells are differentiated into hair cells. Incertain embodiments, differentiation is induced by use of a notchinhibitor. Notch pathway regulators include but are not limited to thoselisted, referenced 69, or disclosed in U.S. Pat. No. 8,377,886, which isincorporated by reference herein in its entirety.

R04929097; DAPT(N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethylester); L-685458((5S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L-leu-L-phe-amide);BMS-708163 (Avagacestat); BMS-299897(2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoicacid); M-0752; YO-01027; MDL28170 (Sigma); LY41 1575(N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yI)-1-alaninamide);ELN-46719 (2-hydroxy-valeric acid amide analog of LY41 1575; PF-03084014((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide);Compound E((2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide;and Semagacestat (LY450139; (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-2-oxoethyl)butanamide),or pharmaceutically acceptable salts thereof.

Administration

The membrane of the round window is the biological barrier to the innerear space and represents the major obstacle for the local treatment ofhearing impairment. The administered drug must overcome this membrane toreach the inner ear space. The drug can operatively (e.g., injectionthrough the tympanic membrane) be placed locally to the round windowmembrane and can then penetrate through the round window membrane.Substances that penetrate the round window typically distribute in theperilymph and thus reach the hair cells and supporting cells.

In certain embodiments, pharmaceutical formulations are adapted toadminister the drug locally to the round window membrane. Thepharmaceutical formulations may also contain a membrane penetrationenhancer, which supports the passage of the agents mentioned hereinthrough the round window membrane. Accordingly, liquid, gel or foamformulations may be used. It is also possible to apply the activeingredient orally or to employ a combination of delivery approaches.

Intratympanic (IT) delivery of drugs to the ear is increasingly used forboth clinical and research purposes. Some groups have applied drugs in asustained manner using microcatheters and microwicks, while the majorityhave applied them as single or as repeated IT injections (up to 8injections over periods of up to 2 weeks) 8.

Intratympanically applied drugs are thought to enter the fluids of theinner ear primarily by crossing the round window (RW) membrane.Calculations show that a major factor controlling both the amount ofdrug entering the ear and the distribution of drug along the length ofthe ear is the duration the drug remains in the middle ear space.Single, ‘one-shot’ applications or applications of aqueous solutions forfew hours' duration result in steep drug gradients for the appliedsubstance along the length of the cochlea and rapidly decliningconcentration in the basal turn of the cochlea as the drug subsequentlybecomes distributed throughout the ear.

Other injection approaches include by osmotic pump, or, by combinationwith implanted biomaterial, and more preferably, by injection orinfusion. Biomaterials that can aid in controlling release kinetics anddistribution of drug include hydrogel materials, degradable materials.One class of materials that is most preferably used includes in situgelling materials. All potential materials and methodologies mentionedin these reference are included herein by reference.^(11,13-58) Othermaterials include collagen or other natural materials including fibrin,gelatin, and decelluarized tissues. Gelfoam may also be suitable.

Delivery may also be enhanced via alternate means including but notlimited to agents added to the delivered composition such as penetrationenhancers, or could be through devices via ultrasound, electroporation,or high speed jet.

Methods described herein can also be used for inner ear cell types thatmay be produced using a variety of methods know to those skilled in theart including those cell types described in PCT Application No.WO2012103012 A1.

With regard to human and veterinary treatment, the amount of aparticular agent(s) that is administered may be dependent on a varietyof factors, including the disorder being treated and the severity of thedisorder; activity of the specific agent(s) employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific agent(s) employed; the duration of the treatment; drugs used incombination or coincidental with the specific agent(s) employed; thejudgment of the prescribing physician or veterinarian; and like factorsknown in the medical and veterinary arts.

The agents described herein may be administered in a therapeuticallyeffective amount to a subject in need of treatment. Administration ofcompositions described herein can be via any of suitable route ofadministration, particularly by intratympanically. Other routes includeingestion, or alternatively parenterally, for example intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrasternally, intracranially, intramuscularly,intranasally, subcutaneously, sublingually, transdermally, or byinhalation or insufflations, or topical by ear instillation forabsorption through the skin of the ear canal and membranes of theeardrum. Such administration may be as a single or multiple oral dose,defined number of ear drops, or a bolus injection, multiple injections,or as a short- or long-duration infusion. Implantable devices (e.g.,implantable infusion pumps) may also be employed for the periodicparenteral delivery over time of equivalent or varying dosages of theparticular formulation. For such parenteral administration, thecompounds are preferably formulated as a sterile solution in water oranother suitable solvent or mixture of solvents. The solution maycontain other substances such as salts, sugars (particularly glucose ormannitol), to make the solution isotonic with blood, buffering agentssuch as acetic, citric, and/or phosphoric acids and their sodium salts,and preservatives. The preparation of suitable, and preferably sterile,parenteral formulations is described in detail in the section entitled“Compositions”, above.

Compositions described herein can be administered by a number of methodssufficient to deliver the composition to the inner ear. Delivering acomposition to the inner ear includes administering the composition tothe middle ear, such that the composition may diffuse across the roundwindow to the inner ear and administering a composition to the inner earby direct injection through the round window membrane. Such methodsinclude, but are not limited to auricular administration, bytranstympanic wicks or catheters, or parenteral administration, forexample, by intraauricular, transtympanic, or intracochlear injection.

In particular embodiments, the compositions and formulations of thedisclosure are locally administered, meaning that they are notadministered systemically.

In one embodiment, a syringe and needle apparatus is used to administercompositions to a subject using auricular administration. A suitablysized needle is used to pierce the tympanic membrane and a wick orcatheter comprising the composition is inserted through the piercedtympanic membrane and into the middle ear of the subject. The device maybe inserted such that it is in contact with the round window orimmediately adjacent to the round window. Exemplary devices used forauricular administration include, but are not limited to, transtympanicwicks, transtympanic catheters, round window microcatheters (smallcatheters that deliver medicine to the round window), and SilversteinMicrowicks™ (small tube with a “wick” through the tube to the roundwindow, allowing regulation by subject or medical professional).

In another embodiment, a syringe and needle apparatus is used toadminister compositions to a subject using transtympanic injection,injection behind the tympanic membrane into the middle and/or inner ear.The formulation may be administered directly onto the round windowmembrane via transtympanic injection or may be administered directly tothe cochlea via intracochlear injection or directly to the vestibularorgans via intravestibular injection.

In some embodiments, the delivery device is an apparatus designed foradministration of compositions to the middle and/or inner ear. By way ofexample only: GYRUS Medical Gmbh offers micro-otoscopes forvisualization of and drug delivery to the round window niche; Arenberghas described a medical treatment device to deliver fluids to innerearstructures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446,each of which is incorporated by reference herein for such disclosure.U.S. patent application Ser. No. 08/874,208, which is incorporatedherein by reference for such disclosure, describes a surgical method forimplanting a fluid transfer conduit to deliver compositions to the innerear. U.S. Patent Application Publication 2007/0167918, which isincorporated herein by reference for such disclosure, further describesa combined otic aspirator and medication dispenser for transtympanicfluid sampling and medicament application.

In some embodiments, a composition disclosed herein is administered toan subject in need thereof once. In some embodiments, a compositiondisclosed herein is administered to an subject in need thereof more thanonce. In some embodiments, a first administration of a compositiondisclosed herein is followed by a second, third, fourth, or fifthadministration of a composition disclosed herein.

The number of times a composition is administered to an subject in needthereof depends on the discretion of a medical professional, thedisorder, the severity of the disorder, and the subject's response tothe formulation. In some embodiments, a composition disclosed herein isadministered once to an subject in need thereof with a mild acutecondition. In some embodiments, a composition disclosed herein isadministered more than once to an subject in need thereof with amoderate or severe acute condition. In the case wherein the subject'scondition does not improve, upon the doctor's discretion the compositionmay be administered chronically, that is, for an extended period oftime, including throughout the duration of the subject's life in orderto ameliorate or otherwise control or limit the symptoms of thesubject's disease or condition.

In the case wherein the subject's status does improve, upon the doctor'sdiscretion the composition may administered continuously; alternatively,the dose of drug being administered may be temporarily reduced ortemporarily suspended for a certain length of time (i.e., a “drugholiday”). The length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dosereduction during a drug holiday may be from 10%-100%, including by wayof example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once the subject's hearing and/or balance has improved, a maintenancedose can be administered, if necessary. Subsequently, the dosage or thefrequency of administration, or both, is optionally reduced, as afunction of the symptoms, to a level at which the improved disease,disorder or condition is retained. In certain embodiments, subjectsrequire intermittent treatment on a long-term basis upon any recurrenceof symptoms.

Formulations

The biologically active compositions described herein (sometimesreferred to herein as “biologically active agents” or more simply, as“agents”) may be formulated in any manner suitable for a desireddelivery route to an in vitro population or to an in vivo population ofcells, e.g., transtympanic injection, transtympanic wicks and catheters,and injectable depots. Typically, such formulations include allphysiologically acceptable forms of the biologically activecompositions, including free acid forms, free base forms, acid additionsalts, base addition salts, other derivative thereof (such as a prodrugor solvate thereof), racemic, optically active, or tautomer thereof withany physiologically acceptable carriers, diluents, and/or excipients.

Solid formulations of the compositions described herein, such asdragees, capsules, pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings. Solid dosage forms may also be formulated so as to provideslow or controlled release of the ion channel modulating compound. Thus,solid formulations could include any material that could provide adesired release profile of the ion channel modulating compound,including but not limited to hydroxypropylmethyl cellulose in varyingproportions, or other polymer matrices, liposomes and/or microspheres.

Pharmaceutically acceptable carriers can include but are not limited to:water, ethanol, oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, and the like, an excipient such as methylcellulose, carageenan, andthe like.

Liquid dosage formulations may include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition, the liquid dosage formulations may contain inert diluentscommonly used in the art, including but not limited to water or othersolvents; solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol; oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils); glycerol; tetrahydrofuryl alcohol; polyethylene glycols; andfatty acid esters of sorbitan, and mixtures thereof.

Suspensions formulations include, without limitation, ethoxylatedisostearyl alcohols; polyoxyethylene sorbitol and sorbitan esters;microcrystalline cellulose; aluminum metahydroxide; bentonite;agar-agar; tragacanth; and mixtures thereof.

Proper fluidity of liquid, suspension and other formulations of the ionchannel modulating compounds can be maintained by the use of coatingmaterials such as lecithin; by the maintenance of the required particlesize in the case of dispersions; or by the use of surfactants.

Formulations may also include anti-contamination agents for theprevention of microorganism contamination. Anti-contamination agents mayinclude but are not limited to antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, antibiotics, andthe like.

Formulations may also be sterilized by, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid formulations which can be dissolvedin sterile water, or some other sterile medium immediately before use orformulation.

Formulations may also be endotoxin free. As used herein, the term“endotoxin free” refers to compositions or formulations that contain atmost trace amounts (i.e., amounts having no adverse physiologicaleffects to a subject) of endotoxin, and preferably undetectable amountsof endotoxin. By “substantially free of endotoxin” is meant that thereis less endotoxin per dose of cells than is allowed by the FDA for abiologic, which is a total endotoxin of 5 EU/kg body weight per day,which for an average 70 kg person is 350 EU per total dose of cells. Inone embodiment, the term “endotoxin free” refers to a composition orformulation that is at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% endotoxin free. Endotoxins are toxinsassociated with certain bacteria, typically gram-negative bacteria,although endotoxins may be found in gram-positive bacteria, such asListeria monocytogenes. The most prevalent endotoxins arelipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in theouter membrane of various Gram-negative bacteria, and which represent acentral pathogenic feature in the ability of these bacteria to causedisease. Small amounts of endotoxin in humans can produce fever, alowering of the blood pressure, and activation of inflammation andcoagulation, among other adverse physiological effects. Therefore, it isoften desirable to remove most or all traces of endotoxin from drugproduct containers, because even small amounts may cause adverse effectsin humans.

Pharmaceutical compositions described herein are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a subject. For example,pharmaceutical compositions described herein can be prepared bycombining a Notch Activator and/or HDAC inhibitor and a GSK3b inhibitorand/or WNT activator and/or small molecules with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid, gels, andmicrospheres, including those formulations adapted for auricularadministration, by transtympanic wicks or catheters, or parenteraladministration, for example, by intraauricular, transtympanic, orintracochlear injection. However, in certain embodiments the subjectcompounds may be simply dissolved or suspended in sterile water.

Coated, gel, or encapsulating formulations of a Notch Activator and/orHDAC inhibitor and a GSK3b inhibitor and/or WNT activator or derivativesand/or small molecules may also be formulated to deliver pulsatile,sustained, or extended release. For example, one method of pulsatilerelease could be achieved by layering multiple coatings of agents orderivatives and/or small molecules, or by incorporating the agentsderivatives and/or small molecules within different regions of theformulation having different release times.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the composition in biodegradable polymers. Examples ofbiodegradable polymers include, but are not limited topolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Theratio of composition to polymer and the nature of the particular polymeremployed can affect the rate of release of Notch Activators and/or HDACinhibitor and a GSK3b inhibitor and/or WNT activators, or derivativesand/or small molecules from the composition. Depot injectableformulations can also be prepared by entrapping the drug in liposomes ormicroemulsions.

Pharmaceutical compositions may further comprise one or more componentsthat enhance the bioavailability of the active ingredients of thecomposition, e.g., penetration enhancers, stabilizing agents, and one ormore components that provide slow or controlled release of the NotchActivator and/or HDAC inhibitor and a GSK3b inhibitor and/or WNTactivator derivatives and/or small molecules in the composition, e.g.,biocompatible polymers and/or gels.

In particular embodiments, compositions comprising penetration enhancerswill facilitate the delivery of the composition across biologicalbarriers that separate the middle and inner ear, e.g., the round window,thereby efficiently delivery a therapeutically effective amount of thecomposition to the inner ear. Efficient delivery to the cochlea, Organof Corti, and/or vestibular organs is desired because these tissues hostthe support cells that promote sensory hair cell regeneration whentreated or contacted with compositions described herein.

A “penetration enhancer” or “permeability enhancer” includes a polyolsuch as polyethylene glycol (PEG), glycerol (glycerin), maltitol,sorbitol etc.; diethylene glycol monoethyl ether, azone, benzalkoniumchloride (ADBAC), cetylperidium chloride, cetylmethylammonium bromide,dextran sulfate, lauric acid, menthol, methoxy salicylate, oleic acid,phosphatidylcholine, polyoxyethylene, polysorbate 80, sodium glycholate,sodium lauryl sulfate, sodium salicylate, sodium taurocholate, sodiumtaurodeoxycholate, sulfoxides, sodium deoxycholate, sodiumglycodeoxycholate, sodium taurocholate and surfactants such as sodiumlauryl sulfate, laureth-9, cetylpyridinium chloride and polyoxyethylenemonoalkyl ethers, benzoic acids, such as sodium salicylate and methoxysalicylate, fatty acids, such as lauiic acid, oleic acid, undpcanoicacid and methyl oleate, fatty alcohols, such as octanol and nonanol,laurocapram, cyclodextrins, thymol, limonene, urea, chitosan and othernatural and synthetic polymers.

Other penetration enhancers include but are not limited to thosedescribed in US patent application publication number: US20110166060.

Suitable polyols for inclusion in the solutions described herein includeglycerol and sugar alcohols such as sorbitol, mannitol or xylitol,polyethylene glycol and derivatives thereof. In some embodiments thecomposition further includes a preservative. Accepted preservatives suchas benzalkonium chloride and disodium edetate (EDTA) are included in thecompositions described herein in concentrations sufficient for effectiveantimicrobial action, about 0.0001 to 0.1%, based on the weight of thecomposition.

In particular embodiments, compositions of the present disclosure alsoinclude stabilizers to increase the therapeutic lifetime of thecompositions in vivo. Exemplary stabilizers include fatty acids, fattyalcohols, alcohols, long chain fatty acid esters, long chain ethers,hydrophilic derivatives of fatty acids, polyvinyl pyrrolidones,polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobicpolymers, moisture-absorbing polymers, and combinations thereof. Infurther embodiments, the chosen stabilizer changes the hydrophobicity ofthe formulation (e.g., oleic acid, waxes), or improves the mixing ofvarious components in the formulation (e.g., ethanol), affects themoisture level in the formula (e.g., PVP or polyvinyl pyrrolidone),affects the mobility of the phase (substances with melting points higherthan room temperature such as long chain fatty acids, alcohols, esters,ethers, amides etc. or mixtures thereof; waxes), and/or improves thecompatibility of the formula with encapsulating materials (e.g., oleicacid or wax). In other embodiments, stabilizers are present insufficient amounts to inhibit the degradation of the Notch Activatorand/or HDAC inhibitor and a GSK3b inhibitor and/or WNT activatorderivatives and small molecules in the composition. Examples of suchstabilizing agents, include, but are not limited to: (a) about 0.5%> toabout 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c)about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% toabout 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k)cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m)divalent cations such as magnesium and zinc; or (n) combinationsthereof.

In particular embodiments, compositions of the disclosure are formulatedas controlled release formulations. In general, controlled release drugformulations impart control over the release of drug with respect tosite of release and time of release in vivo. Controlled release includesto immediate release, delayed release, sustained release, extendedrelease, variable release, pulsatile release and bi-modal release.

Advantages offered by controlled release include: less frequent dosing;more efficient drug utilization; localized drug delivery by placement ofa delivery device or formulation at a treatment site in vivo; and theopportunity to administer and release two or more different drugs, eachhaving a unique release profile, or to release the same drug atdifferent rates or for different durations, by means of a single dosageunit.

Controlled release formulations may be made by formulating thecompositions with biocompatible polymers, viscosity agents, gels,paints, foams, xerogels, microparticles, hydrogels, nanocapsules, andthermoreversible gels, or combinations thereof. In certain embodiments,the polymer or gels are biodegradable. Release properties are oftencontrolled by the particular combination of polymers or gels used toformulate the composition. These methods are well known in the art.

Exemplary polymers suitable for formulating the biologically activecompositions of the present disclosure include, but are not limited topolyamides, polycarbonates, polyalkylenes (polyethylene glycol (PEG)),polymers of acrylic and methacrylic esters, polyvinyl polymers,polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof,celluloses, polypropylene, polyethylenes, polystyrene, polymers oflactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone),polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, andblends, mixtures, or copolymers thereof.

In one embodiment, a biologically active composition of the presentdisclosure is formulated in a ABA-type or BAB-type triblock copolymer ora mixture thereof, wherein the A-blocks are relatively hydrophobic andcomprise biodegradable polyesters or poly(orthoester), and the B-blocksare relatively hydrophilic and comprise polyethylene glycol (PEG). Thebiodegradable, hydrophobic A polymer block comprises a polyester orpoly(ortho ester), in which the polyester is synthesized from monomersselected from the group consisting of D,L-lactide, D-lactide, L-lactide,D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid,ε-caprolactone, ε-hydroxyhexanoic acid, γ-butyrolactone,γ-hydroxybutyric acid, δ-valerolactone, δ-hydroxyvaleric acid,hydroxybutyric acids, malic acid, and copolymers thereof.

Exemplary viscosity agents suitable for use in formulating compositionsdescribed herein include, but are not limited to, hydroxypropylmethylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone,carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate,sodium hyaluronate, acacia (gum arabic), agar, agarose, aluminummagnesium silicate, sodium alginate, sodium stearate, bladderwrack,bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose,microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylatedchitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guargum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol,honey, maize starch, wheat starch, rice starch, potato starch, gelatin,sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose,ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose,hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin,polygeline, povidone, propylene carbonate, methyl vinyl ether/maleicanhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate),poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose,hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose(CMC), silicon dioxide, or polyvinylpyrrolidone (PVP: povidone).

Suitable gelling agents for use in preparation of the gel formulationinclude, but are not limited to, celluloses, cellulose derivatives,cellulose ethers (e.g., carboxymethylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose),guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid),silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan,paraffin, petrolatum and any combinations or mixtures thereof.

A variety of suitable biocompatible polymers can be used for thedelivery of compounds to the ear. Preferably, the polymers can form agel. Examples of suitable gel forming biocompatible polymers includehyaluronic acid, hyaluronates, lecithin gels, (poly)alanine derivatives,pluronics, poly(ethyleneglycol), poloxamers, chitosans, xyloglucans,collagens, fibrins, polyesters, poly(lactides), poly(glycolide) or theirco-polymers PLGA, sucrose acetate isobutyrate, and glycerol monooleate.

Hyaluronic acid is a naturally-occurring, biocompatible polysaccharidethat binds water and forms a degradable gel with high viscosity.Polyethylene glycol (PEG) is a biocompatible polymer, hydrophilicpolymer.

Thermosetting polymers that are fluids at low temperature, but are moreviscous at higher temperatures, are also suitable. Common reversiblethermosetting systems are poloxamers. When dissolved, the solutions canremain liquid at low temperatures, but can form more viscous, solid-likeimplants when the temperature increases.

Chitosan is a biocompatible and has antibacterial properties, and achitosan-glycerolphosphate solution is able to form a reversiblethermosetting gel.

The gel may be also formed from an enzymatically degradable polypeptidepolymer. The polypeptide bonds in the polypeptide polymer are morestable against hydrolysis than the ester bonds in PEG/PLGA polymersystems, and the polypeptide can also include a biodegradable polymerhaving a biodegradable polypeptide block linked to a second polymerblock to form a graft or linear polymer. An example for a polypeptidepolymer is poly(alanine) and derivatives thereof. The polypeptidecarrier may also be a protein matrix known as fibrin. Fibrinogen is anaturally occurring protein which, when combined with the enzymethrombin, another naturally occurring protein, forms a bio-matrix knownas fibrin.

Other biocompatible polymers include starches, celluloses,gelatin-pluronics, tetronics, the latter two being poly(ethyleneoxide)/poly(propylene oxide) materials. Other materials that may be usedinclude the chondroitin sulfates and the general class ofmucopolysaccharides (e.g., glycosaminoglycans) and other biocompatiblepolymers having characteristics similar to hyaluronic acid.

In some instances, the biocompatible polymer may be cross linked.Various cross linking agents for biodegradable materials are known inthe art. Preferably, cross linking is accomplished so that the finalcross linked material for the delivery unit are substantially non-toxic(e.g., by use of thermal cross linking, gamma irradiation, ultravioletirradiation, chemical cross linking, etc.). In general, the degree ofcross linking relates inversely to the degree of swelling or absorptionof water by the shaped polymer structure. The degree of swelling orwater absorption regulates the rate of drug transport by the polymerstructure.

In some embodiments, the drug is administered across the eardrum, e.g.,by piercing, injection, etc. In some embodiments, the drug isadministered across the eardrum without piercing it, e.g., absorptionacross eardrum.

In some embodiments, it is desirable that the formulation besufficiently viscous that it remain in an area of administration for asuitable period of time, e.g., 1 min, 5 min, 10 min, 30 min., 1 hour, 5hours, 12, hours, 1 day, 2 days, 7 days. The desired area may beexternal to the eardrum, but in contact therewith, e.g., where it can bedesired that a layer of solution remain in contact with the eardrum(e.g., not flow out of ear) when the recipient subject is upright; evenif some of a viscous formulation exits the ear canal, a viscous film oflayer can, in some embodiments, be let in contact with the eardrum.Similarly, in embodiments be administered across the eardum, e.g., by

It should be appreciated that formulations can be altered in someembodiments by adjusting the liquid-gel transition such that it is rapidand reproducible, while loaded with drug.

For one carrier, poloxamer 407, gelation can decrease with increasingthe concentration of poloxamer 407, at least in the absence of drug.Addition of some drugs, e.g., hydrophilic drugs, including VPA and pVc,at concentrations greater than 88 mg/ml and 14 mg/ml, respectively,inhibited gelation of poloxamer-407 solution. Therefore, gels wereprepared using 18% (w/w) poloxamer solutions with concentrations of VPAand pVc to be equal to or less than 88 mg/ml and 14 mg/ml, respectively.Hydrophobic drugs, including CHIR, Repsox and TTNPB, appropriate volumesfrom their stock solutions in DMSO were added into the poloxamer-407solutions containing the hydrophilic drugs, and mixed by pipetting at 4°C. Concentrations of drugs in stock solutions were maintained at55.6-69.5 mg/ml, 23-28.75 mg/ml and 35 mg/ml for CHIR, Repsox and TTNPB,respectively to ensure total DMSO concentration in final formulation tobe less than 5-6%. Higher concentration of DMSO significantly loweredthe gelation temperature of the formulations. The final formulation wasa viscous liquid at storage temperature (4° C.), and formed a semisolidgel above its liquid-gel transition temperature (37° C.).

In some cases, compositions have a viscosity of less than 100,000centipoise (cps) at 25° C. Compositions also have a minimum yield stressthat is sufficient for maintaining the formulation against the tympanicmembrane. Yield stress is the amount of force that causes a solidmaterial to exhibit liquid-like behavior in that it continues to deformwith no further increase in stress. Minimum yield stress is dependent onthe thickness of the applied gel, but is independent of the geometry ofthe gel and the temperature of the environment. Minimum yield stress ofa composition refers to an applied gel having a thickness of 4 mm and adensity of 1 g/L. Yield stress (σ₀) is represented as σ₀=ρgh, where ρ isdensity, g is the acceleration due to gravity, and h is the layerthickness. Typically, minimum yield stress is about 39 pascals (Pa).

Viscogenic agents are typically polymers or other chemical moieties thatincrease the viscosity of a fluid. Suitable viscogenic agents, whenincluded in a composition, allow the composition to transform from aliquid-like state (e.g., flowable) at 25° C. to a solid-like state(e.g., a gel) after contact with the tympanic membrane, and can benon-biodegradable, (e.g., not broken down by chemicals or enzymesnaturally present in a mammal, or biodegradable). Compositions includean amount of viscogenic agent effective to yield a viscosity of thecomposition of less than 100,000 cps at 25° C. (e.g., less than 90,000,60,000, 30,000, 20,000, or 10,000 cps) and, generally, a minimum yieldstress of 39 Pa after application to the tympanic membrane. Typically, acomposition includes 0.05 to 50% of a viscogenic agent (e.g., 0.15 to25, 5 to 45, 10 to 40, 12 to 37, 15 to 35, 17 to 33, or 20 to 30% of aviscogenic agent).

Exemplary viscogenic agents include gellan (GELRITE or KELCOGEL),CARBOPOL 940 with hydroxypropylmethylcellulose (HPMC), N-isopropylacrylamide (NiPAAm) with sodium acrylate and n-N-alkylacrylamide,polyacrylic acid with polyethylene glycol (PEG) or polymethacryhc acidwith PEG, cellulose acetate hydrogen phthalate latex (CAP), sodiumalginate, and nonionic surfactants such as poloxamers (PLURIONIC) andpolyoxamine (TETRONIC) reversible temperature-dependent gelling systems.Gellan is a natural polymer, anionic deacetylated exocellularpolysaccharide, secreted by Pseudomonas elodea. The tetrasacchariderepeating unit consists of one α-L-rhamnose, one β-D-glucuronic acid,and two β-D-glucose moieties. The in situ gelling mechanism of gellan iscation-induced (e.g., presence of calcium ions) andtemperature-dependent (e.g., physiologic temperature). Gelation isthermally reversible. CARBOPOL 940 with HPMC gels in situ in apH-dependent manner. CARBOPOL is the gelling agent and the HPMC is usedto enhance the viscosity of the gel. NiPAAm with sodium acrylate andn-N-alkylacrylamide is a terpolymer hydrogel that can undergo atemperature based reversible sol-gel transformation. Sodium acrylate andn-N-alkylacrylamide are used to modify the properties of the hydrogel,and in particular, the transition temperature.

Polyacrylic acid with PEG or polymethacryhe acid with PEG is thought togel based on hydrogen bonding. Polyacrylic acid can be dissolved inhydroalcoholic solution and after being injected, the alcohol diffusesout causing the polymers to precipitate and gelling of the solution. CAPis a nanoparticulate system that gels in a pH-dependent manner. Theactive compound (pharmacologic agent) is adsorbed partially onto thesurface of the polymer particles. Sodium alginate gels in the presenceof calcium or other polyvalent ion.

Poloxamers are triblock copolymers formed of (i.e., hydrophilicpoly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks)configured as a triblock ofpoly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene). Poloxamers areone class of block copolymer surfactants having a propylene oxide blockhydrophobe and an ethylene oxide hydrophile. Poloxamers are commerciallyavailable (e.g., Pluronic® polyols are available from BASF Corporation).Alternatively, polaxamers can be synthesized by known techniques.

Formulations described herein are suitable for inhalation orintravenous, intraperitoneal, intramuscular, subcutaneous,mucocutaneous, oral, rectal, transdermal, topical, buccal, intradermal,intragastric, intradermal, intranasal, intrabuccal, percutaneous orsublingual administration. In certain embodiments administration is viainjection into the middle ear as well as topical administration to thedrum. Formulations may also be administered directly to the inner ear.Formulations can be administered via multiple vehicles includingbiomaterials, solutions, devices (including but not limited to hearingaids, cochlear implants, headphones, and earbuds).

The pharmaceutical compositions can be prepared and administered in theform of transdermal delivery system (patch film), drops, pills, tablets,film-coated tablets, multilayer tablets, gels, ointments, syrups,granules, suppositories, emulsions, dispersions, microcapsules,nanoparticles, microparticles, capsules, powders or injectablesolutions. Pharmaceutical formulations preferably in form of liposomes,emulsions and gels and combinations thereof.

Embodiments described herein also include pharmaceutical compositions,which were prepared using at least one compound described herein, orsalts thereof.

The pharmaceutical compositions may also contain a pharmacologicallyacceptable carrier, excipient and/or solvent.

Such formulations are suitable for inhalation or intravenous,intraperitoneal, intramuscular, subcutaneous, mucocutaneous, oral,rectal, transdermal, topical, buccal, intradermal, intragastric,intradermal, intranasal, intrabuccal, percutaneous or sublingualadministration. In certain embodiments, administration is via injectioninto the middle ear as well as topical administration to the drum.

The pharmaceutical compositions can be prepared and administered in theform of transdermal delivery system (patch film), drops, pills, tablets,film-coated tablets, multilayer tablets, gels, ointments, syrups,granules, suppositories, emulsions, dispersions, microcapsules,capsules, powders or injectable solutions. Pharmaceutical formulationsmay be in form of liposomes, emulsions and gels.

For preparing suppositories, low melting waxes, fatty acid esters andglycerides can be used. Pharmaceutical compositions for any route ofadministration of beta-carbolines contain a sufficient therapeuticeffect for the amount of ß-carboline and, if necessary, inorganic ororganic, solid or liquid pharmaceutically acceptable carriers.Pharmaceutical compositions which are suitable for enteral or parenteraladministration include tablets or gelatine capsules or aqueous solutionsor suspensions as described above.

The pharmaceutical compositions may be sterilized and/or containadjuvants, such as preservatives, stabilizers, wetting agents and/oremulsifiers, salts for regulating the osmotic pressure and/or buffers.The inventive pharmaceutical composition may, if desired, contain otheractive ingredients. These pharmaceutical compositions may be prepared byany method that is known from the prior art are prepared, for example,by conventional methods such as mixing, granulating, packaging, andlyophilization solution and can range from about 0.01 to 100 percent,preferably between 0, 1, and 50 percent in Lyophilizates contain up to100 percent of 8-carboline.

Certain compositions of the invention for topical administration may beother pharmaceutically acceptable substances and/or substances. Incertain embodiments of the present invention, a topical excipient isselected that does not increase the delivery of beta-carbolines andoptionally further active ingredient or ingredients into the bloodcirculatory system or the central nervous system if it is verbreicht atthe ear, in the ear or in the ear canal. For example, it is may bedesired that the topical excipient does not have a substantial exclusionproperty which enhances percutaneous transmission through the mucosainto the systemic circulatory system. Such carriers includehydro-carboxylic acids, such as anhydrous absorbent hydrophilicpetrolatum (Vaseline) and anhydrous lanolin (for example, Aquaphor), andmeans on the basis of water-oil emulsions such as lanolin and coldcream. Certain embodiments include carriers which are non-exclusivesubstantially and typically include those support materials which arewater soluble, as well as materials based on oil-in-water emulsions(creams or hydrophilic ointments) and substance water-soluble base, suchas for polyethylene glycol-based excipients and aqueous solutions gelledwith various agents were as methyl cellulose, hydroxyethyl cellulose andhydroxpropylmethylcellulose.

All delivery methods and materials mentioned in U.S. Pat. Nos. 5,837,681and 6,593,290 are incorporated herein by reference.

If desired, the composition to be administered may also contain minoramounts of nontoxic auxiliary carrier or excipient substances such aswetting agents, emulsifying agents, or solubilizing agents,antioxidants, antimicrobials, pH buffering agents and the like, forexample, sodium acetate, sodium citrate, cyclodextrin derivatives,sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate,etc. Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington: TheScience and Practice of Pharmacy, Mack Publishing Company, Easton, Pa.,20th Edition, 2000. The composition or formulation to be administeredwill, in any event, contain a quantity of the active compound in anamount effective to alleviate the symptoms of the subject being treated.As those in the art will appreciate, the agents described herein mayalso be formulated for targeted delivery to a subset of tissues or cellsin a subject. In general, targeted delivery is accomplished byformulating a compound of the agents with a targeting moiety. Suchmoieties include lipids, liposomes, and ligands for molecules that bind,or are bound by, other molecules in vivo.

Any derived form of the agents (example synthetic or natural), or aconjugate thereof, can be prepared as an acid salt or as a base salt, aswell as in free acid or free base forms. Such compositions if used toprevent or treat auditory dysfunctions are covered under in certainembodiments described herein.

In some embodiments, the compositions herein also target supportingcells that do not express Lgr5. In certain embodiments, the agentsdescribed herein in addition to promoting the proliferation ofsupporting cells, also impact the differentiation of the supportingcells to cell types that can help enhancing hearing. In some embodimentsthe agents described herein also impact the survival of the supportingcells.

The amount of the agent required for use in treatment may vary not onlywith the particular agent and salt selected, but also with the route ofadministration, the nature of the condition being treated, and the ageand condition of the patient, among other factors, and ultimately isdetermined at the discretion of the attending physician or clinician.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, for example, into a number of discrete, loosely spacedadministrations, such as multiple ingestations of pill doses, or liquiddoses.

Further still, the agents, and their respective acid or base salts, canbe formulated into liquid, preferably aqueous, formulations for storageand administration, as well as dried formulations that may, for example,be used as powders for administration or be reconstituted into liquidform just prior to administration to a subject. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, etc. the particular agent and optional pharmaceuticaladjuvants in an aqueous carrier. Aqueous carriers include water(particularly water for injection into humans), alcoholic/aqueoussolutions, and emulsions and suspensions. Pharmaceutically acceptableaqueous carriers include sterile buffered isotonic saline solutions.Vehicles may include sodium chloride solution, Ringer's dextrose,dextrose, and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, andinert gases and the like. Non-aqueous solvents may also be usedincluding propylene glycol, ethanol, polyethylene glycol, vegetable oilssuch as olive oil, and injectable organic esters such as ethyl oleate.The neutraceutical, pharmaceutical and veterinary compositions of theagents, whether dry or liquid, are preferably can also be formulated fororal administration.

As used herein, paints (also known as film formers) are solutionscomprised of a solvent, a monomer or polymer, an active agent, andoptionally one or more pharmaceutically-acceptable excipients. Afterapplication to a tissue, the solvent evaporates leaving behind a thincoating comprised of the monomers or polymers, and the active agent. Byway of non-limiting example, paints include collodions (e.g., FlexibleCollodion, USP), and solutions comprising saccharide siloxane copolymersand a cross-linking agent. The paints contemplated for use herein, areflexible such that they do not interfere with the propagation ofpressure waves through the ear. Further, the paints may be applied as aliquid (i.e., solution, suspension, or emulsion), a semisolid (i.e., agel, foam, paste, or jelly) or an aerosol.

Examples of suitable foamable carriers for use in the compositionsdisclosed herein include, but are not limited to, alginate andderivatives thereof, carboxymethylcellulose and derivatives thereof,collagen, polysaccharides, including, for example, dextran, dextranderivatives, pectin, starch, modified starches such as starches havingadditional carboxyl and/or carboxamide groups and/or having hydrophilicside-chains, cellulose and derivatives thereof, agar and derivativesthereof, such as agar stabilized with polyacrylamide, polyethyleneoxides, glycol methacrylates, gelatin, gums such as xanthum, guar,karaya, gellan, arabic, tragacanth and locust bean gum, or combinationsthereof. The formulation optionally further comprises a foaming agent,which promotes the formation of the foam, including a surfactant orexternal propellant. Examples of suitable foaming agents includecetrimide, lecithin, soaps, silicones and the like. Commerciallyavailable surfactants such as Tween™ are also suitable.

In particular embodiments, gel formulations that are useful inpracticing methods described herein include, but are not limited to,glycerin-based gels, glycerin-derived compounds, conjugated, orcrosslinked gels, matrices, hydrogels, and polymers, as well as gelatinsand their derivatives, alginates, and alginate-based gels, and variousnative and synthetic hydrogel and hydrogel-derived compounds.

In some embodiments, the compositions described herein have aconcentration of each pharmaceutically active ingredient (i.e., NotchActivator and/or DAC inhibitor and a GSK3b inhibitor and/or WNTactivator or derivatives, small molecules, pharmaceutically acceptablesalts, prodrugs, solvates, stereoisomers, racemates, or tautomersthereof) of between about 0.01% to about 90%>, between about 0.01) toabout 50%), between about 0.1% to about 70%>, between about 0.1% toabout 50%0, between about 0.1% to about 40%>, between about 0.1% toabout 30%, between about 0.1%) to about 20%, between about 0.1% to about10%, or between about 0.1% to about 5%), of the each active ingredient,by weight of the composition.

In some embodiments, the compositions described herein have aconcentration of each active pharmaceutical agent between about 1% toabout 50%, between about 5% to about 50%, between about 10% to about40%, or between about 10% to about 30%, of the active ingredient, orpharmaceutically acceptable salt, prodrug, solvate, stereoisomer,racemate, or tautomer thereof, by weight of the composition.

In some embodiments, the formulations described herein have aconcentration of active pharmaceutical ingredient of between about 0.1to about 70 mg/mL, between about 0.5 mg/mL to about 70 mg/mL, betweenabout 0.5 mg/mL to about 50 mg/mL, between about 0.5 mg/mL to about 20mg/mL, between about 1 mg to about 70 mg/mL, between about 1 mg to about50 mg/mL, between about 1 mg/mL and about 20 mg/mL, between about 1mg/mL to about 10 mg/mL, or between about 1 mg/mL to about 5 mg/mL, ofthe active agent, or pharmaceutically acceptable salt, prodrug, solvate,stereoisomer, racemate, or tautomer thereof, by volume of theformulation.

In one embodiment, the formulations disclosed herein additionallyprovide an immediate release of one or more pharmaceutically activeingredients (i.e., a Notch Activator and/or HDAC inhibitor and a GSK3binhibitor and/or WNT activator or other small molecules,pharmaceutically acceptable salts, prodrugs, solvates, stereoisomers,racemates, or tautomers thereof) from the composition, or within 1minute, or within 5 minutes, or within 10 minutes, or within 15 minutes,or within 30 minutes, or within 60 minutes or within 90 minutes. Inother embodiments, a therapeutically effective amount of at least onepharmaceutically active ingredient (i.e., a Notch Activator and/or HDACinhibitor and a GSK3b inhibitor and/or WNT activator or derivatives,small molecules, pharmaceutically acceptable salts, prodrugs, solvates,stereoisomers, racemates, or tautomers thereof) is released from thecomposition immediately, or within 1 minute, or within 5 minutes, orwithin 10 minutes, or within 15 minutes, or within 30 minutes, or within60 minutes or within 90 minutes.

In other embodiments, the composition is formulated as an extendedrelease formulation. In certain embodiments, diffusion of at least onepharmaceutically active ingredient (including a Notch Activator and/orHDAC inhibitor and a GSK3b inhibitor and/or WNT activator), smallmolecules, pharmaceutically acceptable salts, prodrugs, solvates,stereoisomers, racemates, or tautomers thereof) from the formulationoccurs for a time period exceeding 5 minutes, or 15 minutes, or 30minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days,or 10 days, or 12 days, or 14 days, or 18 days, or 21 days, or 25 days,or 30 days, or 45 days, or 2 months or 3 months or 4 months or 5 monthsor 6 months or 9 months or 1 year. In other embodiments, atherapeutically effective amount of at least one pharmaceutically activeingredient (including a Notch Activator and/or HDAC inhibitor and aGSK3b inhibitor and/or WNT activator) is released from the formulationfor a time period exceeding 5 minutes, or 15 minutes, or 30 minutes, or1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or 1 day, or 2days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days,or 12 days, or 14 days, or 18 days, or 21 days, or 25 days, or 30 days,or 45 days, or 2 months or 3 months or 4 months or 5 months or 6 monthsor 9 months or 1 year.

In further embodiments, the formulation provides both an immediaterelease and an extended release formulation. In particular embodiments,the formulation contains a 0.25:1 ratio, or a 0.5:1 ratio, or a 1:1ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5 ratio, or a1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratio ofimmediate release and extended release formulations. In a furtherembodiment the formulation provides an immediate release of a firstpharmaceutically active ingredient (i.e., small molecules,pharmaceutically acceptable salts, prodrugs, solvates, stereoisomers,racemates, or tautomers thereof) and an extended release of a secondpharmaceutically active ingredient or other therapeutic agent. In someembodiments, the formulation provides a 0.25:1 ratio, or a 0.5:1 ratio,or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratioof immediate release and extended release formulations of a firstpharmaceutically active ingredient and second pharmaceutically activeingredient.

The combination of immediate release, delayed release and/or extendedrelease compositions or formulations may be combined with otherpharmaceutical agents, as well as the excipients, diluents, stabilizers,carrier agents and other components disclosed herein. As such, dependingupon the components of the composition, the thickness or viscositydesired, or the mode of delivery chosen, alternative aspects of theembodiments disclosed herein are combined with the immediate release,delayed release and/or extended release embodiments accordingly

F. Administration

In certain embodiments, pharmaceutical formulations are adapted toadminister the drug locally to the round window membrane. Thepharmaceutical formulations may also contain a membrane penetrationenhancer, which supports the passage of the agents mentioned hereinthrough the round window membrane. Accordingly, in such embodimentsliquid or gel formulations may be used. It is also possible to apply theactive ingredient orally or to employ a combination of deliveryapproaches.

The long-acting formulation that prolongs drug release and/or improvedstability may be in the form of agents mentioned herein complexed, orcovalently conjugated (by reversible or irreversible bonding) to amacromolecule such as a water-soluble polymer selected from PEG andpolypropylene glycol homopolymers and polyoxyethylene polyols, i.e.,those that are soluble in water at room temperature. Alternatively, theagent mentioned herein may be complexed or bound to a polymer toincrease its drug release and/or half-life. Examples of polyethylenepolyols and polyoxyethylene polyols useful for this purpose includepolyoxyethylene glycerol, polyethylene glycol, polyoxyethylene sorbitol,polyoxyethylene glucose, or the like. The glycerol backbone ofpolyoxyethylene glycerol is the same backbone occurring in, for example,animals and humans in mono-, di-, and triglycerides. Agents mentionedherein can be encapsulated in materials and/or conjugated to materials.For delivery of more than one agent, one or more delivery vehicle may beused. The delivery kinetics for multiple agents can be the same ordifferent. For example in one case it may be beneficial to include ashort pulse of one drug and have another be released for a longerduration.

Liquid formulations include solutions, suspensions, emulsions andsprays. For example, injection solutions, water-based or water-propyleneglycol for parenteral injection.

Pharmaceutical compositions suitable for topical administration in themiddle ear include aqueous solutions or suspensions which can beprepared prior to administration in the middle ear, such as in the caseof lyophilized formulations that contain a composition of the presentdisclosure, alone or together with a carrier. The pharmaceuticalcompositions further include gels, which is biodegradable ornon-biodegradable, aqueous or non-aqueous or microspheres are based.Examples of such gels and other suitable materials include poloxamers,Hyaluronate, xyloglucans, chitosan, polyesters, polylactide,polyglycolide and their copolymers PLGA polymer, poly anhydrides, polycaprolactone sucrose and glycerol monooleate.

The pharmaceutical compositions may be sterilized and/or containadjuvants, such as preservatives, stabilizers, wetting agents and/oremulsifiers, salts for regulating the osmotic pressure and/or buffers.The pharmaceutical composition may, if desired, contain other activeingredients in addition to those previously described herein.

These pharmaceutical compositions may be prepared by any method that isknown from the prior art are prepared, for example, by conventionalmethods such as mixing, granulating, packaging, and lyophilizationsolution.

The pharmaceutical composition may also include agents to help withcontrolled delivery and/or excipients described in US patent applicationno. US20110166060.

In certain embodiments, the inventive pharmaceutical composition isformulated for topical administration. Suitable carriers for an otogenicadministration are organic or inorganic substances, which arepharmaceutically acceptable and which do not react with the describedagents, and/or other active compounds, for example saline, alcohols,vegetable oils, benzyl alcohols, Alkylglycole, polyethylene glycols,Glycerintriacetate, gelatin, carbohydrates such as lactose or starch,magnesium oxide (magnesia, chalk), stearate (waxes), talc and petrolatum(Vaseline). The compositions described above can be sterilized and/orcontain auxiliaries such as lubricants, preservatives such asthimerosal, for example, 50 percent by weight), stabilizers and/orwetting agents, emulsifiers, salts for influencing the osmotic pressure,buffer substances, colorants and/or flavorings. These compositions maycontain one or more other active ingredients if necessary. Otogenicinventive compositions may comprise different materials and/orsubstances, including other biologically active substances such asantibiotics, anti-inflammatory agents such as steroids, cortisone,analgesics, antipyrine, benzocaine, procaine, etc.

Intratympanic (IT) delivery of drugs to the ear is increasingly used forboth clinical and research purposes. Some groups have applied drugs in asustained manner using microcatheters and microwicks, while the majorityhave applied them as single or as repeated IT injections (up to 8injections over periods of up to 2 weeks).

Intratympanically applied drugs are thought to enter the fluids of theinner ear primarily by crossing the round window (RW) membrane.Calculations show that a major factor controlling both the amount ofdrug entering the ear and the distribution of drug along the length ofthe ear is the duration the drug remains in the middle ear space.Single, ‘one-shot’ applications or applications of aqueous solutions forfew hours' duration result in steep drug gradients for the appliedsubstance along the length of the cochlea and rapidly decliningconcentration in the basal turn of the cochlea as the drug subsequentlybecomes distributed throughout the ear.

Methods for delivering (including relevant materials and permutationsthereof) and methods studying distribution and kinetics of drugdelivered to the inner ear are known in the art. For example, in oneembodiment a 20% (w/w) stock solution of poloxamer 407 gel (SpectrumChemical MFG Corp., Gardena, Calif., USA) is prepared by slowly addingit to cold 10 m M phosphate-buffered saline at pH 7.4. Other poloxamersor other materials may be used. Additional buffer solution is added toobtain a 17% w/w concentration of poloxamer 407 gel. This solution isliquid when refrigerated or at room temperature but solidifies at bodytemperature. The gel can be tinted blue with a die such as Evans bluedye (50 ppm) and sterilized by filtration. Using aseptic techniques,sterilized micronized dex (pure dex in a crystalline, powder form;Pfizer Inc., Kalamazoo, Mich., USA) is suspended with an appropriateamount of sterile poloxamer 407 solution to obtain a 4.5% solution.Samples of the formulation are stored under refrigeration andre-suspended immediately before administration.

Poloxamer-407 hydrogels were prepared using the “cold-method”. Briefly,a weighed amount of poloxamer-407 was added to 40 ml cold ultra purewater or cold PBS (pH 7.4), and stirred overnight at 4° C. on a magneticstir plate to effect complete solubilization. Multiple concentrations ofpoloxamer-407 solution ranging from 18% (w/w) to 25% (w/w) wereprepared. Hydrophilic drugs, including valproic acid (VPA) andphosphorylated ascorbic acid (PAC) were added to the 5 ml poloxamer-407solution and dissolved at 4° C. on a magnetic stir plate. Weight ratioof poloxamer-407 to the drug was varied to understand the effects ofdrugs on the gelation properties of poloxamer-407, and to determine theoptimal formulation that gels at 37° C. with maximum possible loading ofthe hydrophilic drugs. The gelation temperatures of the formulationswere determined by the “visual tube inversion method”. Briefly, glassvials containing poloxamer 407 solutions, with or without thehydrophilic drugs were placed in a water bath. The temperature wasslowly increased and the temperature at which the solution stoppedflowing on tilting the glass vial was noted as the gelation temperature.

To encapsulate hydrophobic drugs, including CHIR 99021 (CHIR), Repsoxand TTNPB, appropriate volumes from their stock solutions in DMSO wereadded into the poloxamer-407 solutions containing the hydrophilic drugs,and mixed by pipetting at 4° C. Maximum DMSO concentration to be addedwith the hydrophobic drugs was limited to 5-6% (v/v) with respect to thetotal volume of the gel. Higher concentration of DMSO reduced thegelation temperature of the gel. Gelation temperature of the formulationwas determined by the “visual tube inversion method”, as describedbefore.

The biologically active agents of the present disclosure may be preparedas an acid salt or as a base salt, as well as in free acid or free baseforms. The amount of the agent required for use in treatment may varynot only with the particular agent and salt selected, but also with theroute of administration, the nature of the condition being treated, andthe age and condition of the patient, among other factors, andultimately is determined at the discretion of the attending physician orclinician. The desired dose may conveniently be presented in a singledose or as divided doses administered at appropriate intervals.

Further still, the active agents, and their respective acid or basesalts, can be formulated into liquid, preferably aqueous, formulationsfor storage and administration, as well as dried formulations that may,for example, be used as powders for administration or be reconstitutedinto liquid form just prior to administration to a subject. Liquidpharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. the particular agent andoptional pharmaceutical adjuvants in an aqueous carrier. Aqueouscarriers include water (particularly water for injection into humans),alcoholic/aqueous solutions, and emulsions and suspensions.Pharmaceutically acceptable aqueous carriers include sterile bufferedisotonic saline solutions. Vehicles may include sodium chloridesolution, Ringer's dextrose, dextrose, and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, and inert gases and the like. Non-aqueous solvents may also beused including propylene glycol, ethanol, polyethylene glycol, vegetableoils such as olive oil, and injectable organic esters such as ethyloleate.

Measuring Hearing

Hearing can be measured using behavioral and or electrical audiometry.

Tests to diagnose hearing loss in humans may include but are not limitedto:

General screening tests. A doctor may ask to cover one ear at a time tosee how well one hears words spoken at various volumes and how theyrespond to other sounds.

Tuning fork tests. Tuning forks are two-pronged, metal instruments thatproduce sounds when struck. Simple tests with tuning forks can help yourdoctor detect hearing loss. A tuning fork evaluation may also revealwhether hearing loss is caused by damage to the vibrating parts of yourmiddle ear (including your eardrum), damage to sensors or nerves of yourinner ear, or damage to both.

Audiometer tests. During these more-thorough tests conducted by anaudiologist, one wears earphones and hear sounds directed to one ear ata time. The audiologist presents a range of sounds of various tones andasks you to indicate each time you hear the sound. Each tone is repeatedat faint levels to find out when you can barely hear. The audiologistwill also present various words to determine one's hearing ability.

Other tests may include:

Auditory Brainstem Response (ABR) Test or Brainstem Auditory EvokedResponse (BAER) Test that checks the brain's response to sound. Becausethis test does not rely on a person's response behavior, the personbeing tested can be sound asleep during the test.

Otoacoustic Emissions (OAE) is a test that checks the inner ear responseto sound. Because this test does not rely on a person's responsebehavior, the person being tested can be sound asleep during the test.

Behavioral Audiometry Evaluation tests how a person responds to soundoverall. Behavioral Audiometry Evaluation tests the function of allparts of the ear. The person being tested must be awake and activelyrespond to sounds heard during the test.

In the case of children, With the parents' permission, the audiologistwill share the results with the child's primary care doctor and otherexperts, such as:

An ear, nose and throat doctor, also called an otolaryngologist, An eyedoctor, also called an ophthalmologist, A professional trained ingenetics, also called a clinical geneticist or a genetics counselor.

Exemplary Embodiments

The present disclosure further includes the following exemplaryembodiments.

In some embodiments “stem cells” includes progenitor cells.

In some embodiments, an FGF1 agonist is used. In some embodiments thismay replace or be added to the Notch activator and/or HDAC inhibitor. Insome embodiments molecules are selected that activate the FGF1 genepromoter. (e.g FGF-1B)

In certain embodiments, cell populations described herein can bedelivered into the inner ear to populate the inner ear and/or enhancehearing. Cell populations have been previously shown to survivefollowing transplantation into the inner ear as described in this patentCN103361300 A and paper.

“In addition, transplanted into the inner ear of guinea pigs, humanamniotic epithelial cells have been shown to survive for up to threeweeks, and the expression of important proteins to maintain homeostasis(Yuge, 1. et al (2004): 77 (9) 1452-1471).”

In certain embodiments, Lgr5+ cells from the inner ear aredifferentiated using a combination of at least one WNT activator and atleast one NOTCH inhibitor.

In certain embodiments, polyoxyethylene-polyoxypropylene triblockcopolymer or derivatives thereof can be used to deliver molecules orfactors described herein to the middle ear and/or for controlledrelease.

In certain embodiments, methods comprise determining a baseline level ofhearing at one or more frequencies before administering the composition,and a subsequent level of hearing at the same one or more frequenciesafter administering the composition, and administering one or moreadditional doses of the composition until a desired level of hearing atthe one or more frequencies is recovered.

In certain embodiments, the subsequent level of hearing is determinedone week, two weeks, three weeks, one month, two months, three months,four months, six months, and/or twelve months after administering thecomposition.

Methods are presented herein wherein the mammal is a child, adolescentor adult, e.g., above the age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13 years.

Methods are presented herein wherein the mammal is an adult of at least40 years of age, e.g., at least 45, 50, 55, 60, 65, 70 years of age.

Methods are presented herein wherein the composition is delivered to amammal.

Methods are presented herein wherein the mammal is a human.

Use of a cell population is described herein, for use in an ototoxicityassay, for example, use of Lgr5⁺ cells in 96 or 384 well plates (orother plate format) to screen agents to boost generation of supportingcells, or proteins expressed by supporting cells (or to screen foragents to suppress protein expression), and/or their differentiation . .. or to identify agents to generate hair cells for ototoxicity or agentsto boost survival of hair cells that is useful to sustain hearingfollowing damage to hair cells or to sustain hair cells followingregeneration. or to screen specific agents to kill specific cellspopulations (Lgr5⁺ cells or their progeny including hair cells) ifneeded. Also use of Lgr5⁺ cells produced herein to serve as cellpopulation for reprogramming or programming protocols.

In certain embodiments, a gamma-secretase inhibitor in addition to aGSK3b inhibitor is used to promote differentiation of supporting cellsinto hair cells.

Methods are presented herein that expand Lgr5⁺ supporting cells with atleast one wnt activator and at least one notch activator (and/or HDACinhibitor) followed by differentiation to hair cells with at least oneWNT activator (preferable embodiment use CHIR) and one notch inhibitor.

A method of treating a subject who has or is at risk of developinghearing loss or vestibular dysfunction, the method comprising:Identifying a subject who has experienced, or is at risk for developing,hearing loss or vestibular dysfunction; Administering to the ear of thesubject a composition comprising one or more compounds that increase wntexpression or activity and inhibits HDAC (and/or activates NOTCH) in acell in the subject's ear; thereby treating the hearing loss orvestibular dysfunction in the subject.

A method of treating a subject who has or is at risk of developinghearing loss or vestibular dysfunction, the method comprisingidentifying a subject who has experienced, or is at risk for developing,hearing loss or vestibular dysfunction, administering to the ear of thesubject a composition comprising CHIR99021

A method of treating a subject who has or is at risk of developinghearing loss or vestibular dysfunction, the method comprising selectinga subject in need of treatment, obtaining a population of cells capableof differentiating into hair cells, contacting the population of cellsin vitro with an effective amount of a composition comprising one ormore compounds that increase wnt expression or activity and inhibitsHDAC (and/or activates NOTCH) for a time sufficient to induce at leastsome of the cells to express one or more of WNT, or reduce or limitexpression of HDAC and/or express NOTCH or homologues thereof, andadministering the population of cells, or a subset thereof, to thesubject's ear.

In one embodiment, cell populations describe herein can be used toscreen for γ-secretase inhibitors that induce hair cell differentiationfrom inner ear stem cells

In one embodiment, a tube in tympanic membrane is placed for continuousand or repeat dosing into the middle ear.

In a particular embodiment, the composition comprises a biodegradablepolymer.

In another embodiment, the composition comprises one or more smallmolecules that decrease the gene expression of Atoh1.

In one embodiment, the composition comprises one or more small moleculesthat decrease the protein expression of Atoh1.

In another embodiment, the composition comprises one or more smallmolecules that increase the gene expression of Atoh1.

In one embodiment, the composition comprises one or more small moleculesthat increase the protein expression of Atoh1.

In a certain embodiment, the composition comprises one or more smallmolecules that increase the activity of Atoh1 protein.

In a further embodiment, the one or more small molecules that increasegene expression of Atoh 1, increase protein expression of Atoh1, orincrease the activity of Atoh1 protein is selected from the groupconsisting of: CHIR99021, 1-Azakenpaullone, and(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO).

In a particular embodiment, the composition comprises a biodegradablepolymer.

In an additional embodiment, the composition is administered to themiddle ear of the subject.

In another embodiment, the composition is administered onto or adjacentto the round window membrane.

In one embodiment, the composition is administered to the inner ear ofthe subject.

In one particular embodiment, the composition is administered to thecochlea of the subject. In one certain embodiment, the composition isadministered to the Organ of Corti of the subject.

In one additional embodiment, the composition is administered bytranstympanic administration.

In one further embodiment, the composition is administered bytranstympanic wick.

In another embodiment, the composition is administered by transtympaniccatheter.

In yet another embodiment, the composition is administered byintracochlear injection.

In various embodiments, the disclosure contemplates, in part, a methodfor promoting cochlear hair cell regeneration comprising administering,to a middle or inner of a subject, a composition comprising a notchactivator (and or HDAC inhibitor) and wnt activator in an amounteffective and for a time sufficient to promote cochlear hair cellproliferation, thereby promoting cochlear hair cell regeneration.

In one embodiment, the subject has a partial or complete loss of hearingor balance.

In a particular embodiment, the subject has sensorineural hearing lossdue to acute or chronic exposure to ototoxic compounds, acute or chronicexposure to noise, age related hearing loss, a genetic related hearingloss, or has auditory neuropathy.

In a certain embodiment, the subject is at risk of developingsensorineural hearing loss or auditory neuropathy.

Methods and agents described in following references are included herein their entirety.

-   Mammalian cochlear supporting cells can divide and    trans-differentiate into hair cells. White P M, Doetzlhofer A, Lee Y    S, Groves A K, Segil N. Nature. 2006 Jun. 22; 441(7096):984-7.-   Lgr5-positive supporting cells generate new hair cells in the    postnatal cochlea. Bramhall N F, Shi F, Arnold K, Hochedlinger K,    Edge A S. Stem Cell Reports. 2014 Feb. 20; 2(3):311-22. doi:    10.1016/j.stemcr.2014.01.008. eCollection 2014 Mar. 11.-   Notch inhibition induces cochlear hair cell regeneration and    recovery of hearing after acoustic trauma. Mizutari K, Fujioka M,    Hosoya M, Bramhall N, Okano H J, Okano H, Edge A S. Neuron. 2013    Jan. 9; 77(1):58-69. doi: 10.1016/j.neuron.2012.10.032. Erratum in:    Neuron. 2013 Apr. 24; 78(2):403.-   Generation of hair cells in neonatal mice by β-catenin    overexpression in Lgr5-positive cochlear progenitors. Shi F, Hu L,    Edge A S. Proc Natl Acad Sci USA. 2013 Aug. 20; 110(34):13851-6.    doi: 10.1073/pnas.1219952110. Epub 2013 Aug. 5.-   Notch signaling alters sensory or neuronal cell fate specification    of inner earstem cells. Jeon S J, Fujioka M, Kim S C, Edge A S. J    Neurosci. 2011 Jun. 8; 31(23):8351-8. doi:    10.1523/JNEUROSCI.6366-10.2011.-   Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors    in the cochlea. Shi F, Kempfle J S, Edge A S. J Neurosci. 2012 Jul.    11; 32(28):9639-48. doi: 10.1523/JNEUROSCI.1064-12.2012. PMID:-   Spontaneous hair cell regeneration in the neonatal mouse cochlea in    vivo. Cox B C, Chai R, Lenoir A, Liu Z, Zhang L, Nguyen D H,    Chalasani K, Steigelman K A, Fang J, Rubel E W, Cheng A G, Zuo J.    Development. 2014 February; 141(4):816-29. doi: 10.1242/dev.103036.

Those skilled in the art will realize that there are many permutationsto how agents described herein or their derivatives may be used anddelivered. Combinations with other agents can also be employed. Relevantpatent references describing other agents that may be used incombination with agents described herein but not limited to (anddescribing methodologies that can be used): US20130324594 A1,WO2011079841 A1, U.S. Pat. No. 7,498,031 B2, U.S. Pat. No. 6,177,434 B1,CA2268331 C, WO2008010852 A2, U.S. Pat. No. 7,387,614 B2, WO1996040094A1, WO2008076556 A2.

Additional Background Information

Auditory Hair Cells in Birds

Currently, hearing loss in mammals is permanent. While frogs, fish, andbirds6 with hearing loss regain their hearing naturally, mammals lostthat ability as much as 300 million years ago, and so far scientistshave been unsuccessful in solving that problem. The ultimate goal ofhair cell regeneration is to restore functional hearing. Because birdsbegin perceiving and producing song early in life, they provide apropitious model for studying not only whether regeneration of lost haircells can return auditory sensitivity but also whether this regeneratedperiphery can restore complex auditory perception and production. Theyare a rare example of where hair cell regeneration occurs naturallyafter hair cell loss and where the ability to correctly perceive andproduce complex acoustic signals is critical to procreation andsurvival. Thus the biology exists for regeneration of the inner earcells to enhance or restore hearing.

Importantly, acoustic overstimulation can lead to sensory cell (haircell) loss in the auditory epithelium. Damaged hair cells in the organof Corti (the mammalian auditory end-organ) degenerate and are replacedby non-sensory cells (supporting cells) which construct an irreversiblescar. In birds, however, auditory hair cells which are damaged byacoustic trauma or ototoxic drugs may be replaced by new hair cells.Supporting cells in damaged regions of the avian auditory epitheliumincorporate the DNA-specific marker bromodeoxyuridine as early as oneday after noise exposure7. Following acoustic insult to the avian innerear, supporting cells which reside within the sensory epithelium dividenear the luminal surface and repopulate the epithelium. These resultssuggest that supporting cells participate in scar formation during haircell degeneration, and produce new cells for regeneration.

Hearing Loss and Destruction of Hair Cells

Hair cells of the inner ear are critical to hearing and vestibularfunction. In mammals, the loss of sensory hair cells is permanent, asthere is no significant capacity for regeneration of these cells. Drugssuch as aminoglycoside antibiotics and many anti-neoplastic drugs areoften used despite unfortunate side effects. One such side effect ishearing loss due to death of the sensory hair cells of the inner ear.Aminoglycosides are clinically used drugs that cause dose-dependentsensorineural hearing loss (Smith et al., New Engl J Med. (1977)296:349-53) and are known to kill hair cells in the mammalian inner ear(Theopold, Acta Otolaryngol (1977) 84:57-64). In the U.S. over 2,000,000people receive treatment with aminoglycosides per year. The clinicalefficacy of these drugs in treating resistant bacterial infections andtheir low cost globally account for their continued use and need.Cisplatin, a chemotherapeutic agent, is also used for its benefit tolife despite its toxic effects on the hair cells of the inner ear. Highfrequency hearing loss (>8 kHZ) has been reported to be as high as 90%in children undergoing cisplatin therapy (Allen, et al., OtolaryngolHead Neck Surg (1998) 118:584-588). The incidence of vestibulotoxiceffects of such drugs on patient populations has been less well studied.Estimates range between 3% and 6% with continued reports in theliterature of patients with aminoglycoside induced vestibulotoxicity(Dhanireddy et al., Arch Otolarngol Head Neck Surg (2005) 131:46-48).Other clinically important and commonly used drugs also have documentedototoxic effects, including loop diuretics (Greenberg, Am J Med Sci.(2000) 319:10-24) and antimalarial quinines (Claessen, et al., Trop MedInt Health, (1998) 3:482-9) salicylates (Matz, Ann Otol Rhinol LaryngolSuppl (1990) 148:39-41).

Research in the past few decades has uncovered some of the keyintracellular events that can cause hair cell death. Several candidateprotectants have been evaluated such as antioxidants, caspaseinhibitors, and jun kinase inhibitors (Kopke R D, et al. Am J Otol 1997,18:559-571; Liu W, et al. Neuroreport 1998, 9:2609-2614; Yamasoba T. etal. Brain Res 1999, 815:317-325: Matsui J I, et al. J Neurosci 2002,22:1218-1227; Sugahara K, et al. Hear Res 2006, 221:128-135.) A few ofthese candidate otoprotectants have progressed to human trials (Sha S H,et al. N Engl J Med 2006, 354:1856-1857; Campbell K C, et al. Hear Res2007, 226:92-103). Further, different cell death pathways may betriggered in response to different forms of damage, and many protectivemolecules offer incomplete hair cell protection, hinting thatpolypharmacy approaches may offer the greatest benefit. Several examplesof agents being explored to protect hair cells are included in US PatentApplication Publication No. US20110135756 A1.

Hearing loss or impairment is a common occurrence for mammals Impairmentanywhere along the auditory pathway from the external auditory canal tothe central nervous system may result in hearing loss. Auditoryapparatus can be divided into the external and middle ear, inner ear andauditory nerve and central auditory pathways. While having somevariations from species to species, the general characterization iscommon for all mammals. Auditory stimuli are mechanically transmittedthrough the external auditory canal, tympanic membrane, and ossicularchain to the inner ear. The middle ear and mastoid process are normallyfilled with air. Disorders of the external and middle ear usuallyproduce a conductive hearing loss by interfering with this mechanicaltransmission. Common causes of a conductive hearing loss includeobstruction of the external auditory canal, as can be caused by auralatresia or cerumen, thickening or perforation of the tympanic membrane,as can be caused by trauma or infection, fixation or resorption of thecomponents of the ossicular chain, and obstruction of the Eustachiantube, resulting in a fluid-filled middle-ear space. Auditory informationis transduced from a mechanical signal to a neurally conductedelectrical impulse by the action of neuro-epithehal cells (hair cells)and SGN in the inner ear. All central fibers of SGN form synapses in thecochlear nucleus of the pontine brain stem. The auditory projectionsfrom the cochlear nucleus are bilateral, with major nuclei located inthe inferior col culus, medial geniculate body of the thalamus, andauditory cortex of the temporal lobe. The number of neurons involved inhearing increases dramatically from the cochlea to the auditory brainstem and the auditory cortex. All auditory information is transduced bya limited number of hair cells, which are the sensory receptors of theinner ear, of which the so-called inner hair cells, numbering acomparative few, are critically important, since they form synapses withapproximately 90 percent of the primary auditory neurons. By comparison,at the level of the cochlear nucleus, the number of neural elementsinvolved is measured in the hundreds of thousands. Thus, damage to arelatively few cells in the auditory periphery can lead to substantialhearing loss. Hence, many causes of sensorineural loss can be ascribedto lesions in the inner ear. This hearing loss can be progressive. Inaddition, the hearing becomes significantly less acute because ofchanges in the anatomy of the ear as the animal ages.

During embryogenesis, the vestibular ganglion, spiral ganglion, and theotic vesicle are derived from the same neurogenic ectoderm, the oticplacode. The vestibular and auditory systems thus share manycharacteristics including peripheral neuronal innervations of hair cellsand central projections to the brainstem nuclei. Both of these systemsare sensitive to ototoxins that include therapeutic drugs,antineoplastic agents, contaminants in foods or medicines, andenvironmental and industrial pollutants. Ototoxic drugs include thewidely used chemotherapeutic agent cisplatin and its analogs (Fleischmanet al, 1975; Stadnicki et al., 1975; Nakai et al., 1982; Berggren etal., 1990; Dublin, 1976; Hood and Berlin, 1986), commonly usedaminoglycoside antibiotics, e.g. gentamicin, for the treatment ofinfections caused by Gram-negative bacteria, (Sera et al., 1987;Hinojosa and Lerner, 1987; Bareggi et al., 1990), quinine and itsanalogs, salicylate and its analogs, and loop-diuretics.

The toxic effects of these drugs on auditory cells and spiral ganglionneurons are often the limiting factor for their therapeutic usefulness.For example, antibacterial aminoglycosides such as gentamicins,streptomycins, kanamycins, tobramycins, and the like are known to haveserious toxicity, particularly ototoxicity and nephrotoxicity, whichreduce the usefulness of such antimicrobial agents (see Goodman andGilman's The Pharmacological Basis of Therapeutics, 6th ed., A. GoodmanGilman et al., eds; Macmillan Publishing Co., Inc., New York, pp.1169-71 (1980) or most recent edition). Aminoglycoside antibiotics aregenerally utilized as broad spectrum antimicrobials effective against,for example, gram-positive, gram-negative and acid-fast bacteria.Susceptible microorganisms include Escherichia spp., Hemophilus spp.,Listeria spp., Pseudomonas spp., Nocardia spp., Yersinia spp.,Klebsiella spp., Enterobacter spp., Salmonella spp., Staphylococcusspp., Streptococcus spp., Mycobacteria spp., Shigella spp., and Serratiaspp. Nonetheless, the aminoglycosides are used primarily to treatinfections caused by gram-negative bacteria and, for instance, incombination with penicillins for the synergistic effects. As implied bythe generic name for the family, all the aminoglycoside antibioticscontain aminosugars in glycosidic linkage. Otitis media is a term usedto describe infections of the middle ear, which infections are verycommon, particularly in children. Typically antibiotics are systemicallyadministered for infections of the middle ear, e.g., in a responsive orprophylactic manner. Systemic administration of antibiotics to combatmiddle ear infection generally results in a prolonged lag time toachieve therapeutic levels in the middle ear, and requires high initialdoses in order to achieve such levels. These drawbacks complicate theability to obtain therapeutic levels and may preclude the use of someantibiotics altogether. Systemic administration is most often effectivewhen the infection has reached advanced stages, but at this pointpermanent damage may already have been done to the middle and inner earstructure. Clearly, ototoxicity is a dose-limiting side-effect ofantibiotic administration. For example, nearly 75% of patients given 2grams of streptomycin daily for 60 to 120 days displayed some vestibularimpairment, whereas at 1 gram per day, the incidence decreased to 25%(U.S. Pat. No. 5,059,591). Auditory impairment was observed from 4 to15% of patients receiving 1 gram per day for greater than 1 week developmeasurable hearing loss, which slowly becomes worse and can lead tocomplete permanent deafness if treatment continues. Ototoxicity is alsoa serious dose-limiting side-effect for cisplatin, a platmumcoordination complex, that has proven effective on a variety of humancancers including testicular, ovarian, bladder, and head and neckcancer. Cisplatin damages auditory and vestibular systems (Fleischman etal, 1975, Stadnicki et al, 1975, Nakai et al, 1982, Carenza et al, 1986,Sera et al, 1987, Bareggi et al, 1990) Sa cylates, such as aspirin, arethe most commonly used therapeutic drugs for their anti-inflammatory,analgesic, anti-pyretic and anti-thrombotic effects. Unfortunately, theyhave ototoxic side effects They often lead to tinnitus (“ringing in theears”) and temporary hearing loss (Myers and Bernstein, 1965). However,if the drug is used at high doses for a prolonged time, the hearingimpairment can become persistent and irreversible, as reportedclinically (Jarvis, 1966)

Pertinent Biology

Fish and birds generate inner ear hair cells where supporting cells inthe cochlea serve as precursor cells. It is believed that hair cellregeneration occurs by two methods: direct transdifferentiation, wheresupporting cells directly become hair cells; and mitotic regeneration,in which supporting hair cells divide and one or both of the resultingcells develops into a hair cell.

It has been established that hair cell regeneration in the auditory andvestibular systems does occur in chickens and other non-mammals, thoughnot in humans. This spontaneous regeneration leads to restoration ofhearing and balance.

Additional Embodiments and Related Aspects

1. A method of facilitating the generation of inner ear hair cells, themethod comprising:

-   -   administering or causing to be administered to a stem cell        population a first composition comprising (i) and (ii): (i) a        GSK3-beta inhibitor or derivative or a pharmaceutically        acceptable salt thereof and/or Wnt agonist or derivative or a        pharmaceutically acceptable salt thereof, and

(ii) a notch agonist or derivative or a pharmaceutically acceptable saltthereof and/or HDAC inhibitor or derivative or a pharmaceuticallyacceptable salt thereof, to expand the stem cell population; and

-   -   exposing the expanded stem cell population to a second        composition comprising GSK3-beta inhibitor and/or a Wnt agonist,        and, optionally, a notch inhibitor, thereby facilitating        generation of inner ear hair cells from the expanded population        of stem cells.

1A. The method of embodiment 1 wherein the stem cell population is an invitro stem cell population.

1B. The method of embodiment 1 wherein the stem cell population is an exvivo stem cell population.

1C. The method of embodiment 1 wherein the stem cell population is an invivo stem cell population.

1D. The method of embodiment 1 wherein the stem cell population is an invivo stem cell population comprised by a subject and the firstcomposition is administered to the stem cell population byadministration of the first composition to the subject.

1E. The method of any of the preceding embodiments wherein the firstcomposition comprises a Wnt agonist.

1F. The method of any of the preceding embodiments wherein the firstcomposition comprises a GSK3-beta inhibitor.

1G. The method of any of the preceding embodiments wherein the firstcomposition comprises a notch agonist.

1H. The method of any of the preceding embodiments wherein the firstcomposition comprises a HDAC inhibitor.

1I. The method of any of the preceding embodiments wherein the secondcomposition comprises a Wnt agonist.

1J. The method of any of the preceding embodiments wherein the secondcomposition comprises a GSK3-beta inhibitor.

1K. The method of any of the preceding embodiments wherein the secondcomposition comprises a notch agonist.

1L. The method of any of the preceding embodiments wherein the secondcomposition comprises a HDAC inhibitor.

1M. The method of any of the preceding embodiments wherein the first andsecond compositions are the same composition.

1N. The method of embodiment 1 or any of embodiments 1A-1L wherein thefirst and second compositions are different compositions.

2. The method of any of the preceding embodiments, wherein the stem cellpopulation comprises supporting cells.

3. The method of embodiment 2, wherein the supporting cells are Lgr5+cells.

4. The method of any of the preceding embodiments, wherein the stem cellpopulation comprises post-natal cells.

5. The method of any of the preceding embodiments, wherein the haircells are inner ear hair cells.

6. The method of any of the preceding embodiments, wherein the haircells are outer ear hair cells.

7. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population a notch agonist that is also an HDACinhibitor.

8. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population a notch agonist that comprises a syntheticmolecule.

9. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population VPA (e.g., in a pharmaceutically acceptableform (e.g., salt)) (e.g., where VPA is a notch agonist that is also anHDAC inhibitor).

10. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population a Wnt agonist that is also a GSK3-betainhibitor.

11. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population a Wnt agonist that comprises a syntheticmolecule.

12. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population CHIR99021 (e.g., in a pharmaceuticallyacceptable form (e.g., salt)) (e.g., where CHIR99021 is a GSK3-betainhibitor.

13. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population a notch inhibitor.

14. The method of embodiment 13, wherein the notch inhibitor comprisesDAPT (e.g., in a pharmaceutically acceptable form (e.g., salt)).

15. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population: (i) CHIR99021 (e.g., in a pharmaceuticallyacceptable form (e.g., salt)) and (ii) VPA (e.g., in a pharmaceuticallyacceptable form (e.g., salt)) (e.g., where (i) comprises CHIR99021 and(ii) comprises VPA).

16. The method of embodiment 15, wherein the administering step furthercomprises administering or causing to be administered to the stem cellpopulation DAPT (e.g., where DAPT is a notch inhibitor).

17. The method of any of the preceding embodiments, wherein theadministering step is carried out by performing one or more injectionsinto the ear (e.g., transtympanically).

18. The method of embodiment 17, wherein the one or more injections areinto the middle ear.

19. The method of embodiment 17, wherein the one or more injections areinto the inner ear.

20. The method of embodiment 17, wherein performing the one or moreinjections comprises anesthetizing the tympanic membrane and/orsurrounding tissue, placing a needle through the tympanic membrane intothe middle ear, and injecting one or both of (i) and (ii).

21. The method of any of the preceding embodiments, wherein theadministering step comprises administering or causing to be administeredto the stem cell population one or more additional agents (e.g., inaddition to (i) and (ii)).

22. The method of embodiment 21, wherein the one or more additionalagents comprises an ROS inhibitor.

23. The method of embodiment 21 or 22, wherein the one or moreadditional agents comprises vitamin C or a derivative thereof.

24. The method of any of embodiments 21 to 23, wherein the one or moreadditional agents comprises a TGFβ type I receptor inhibitor.

25. The method of any of the preceding embodiments, wherein the expandedpopulation of stem cells is at least 3 times larger than the stem cellpopulation prior to the administering step.

26. The method of any of the preceding embodiments, wherein theadministering step comprises administering the notch agonist and/or HDACinhibitor in a pulsatile manner and administering the GSK3-betainhibitor and/or Wnt agonist in a sustained manner.

27. The method of any of the preceding embodiments, wherein the stemcell population is of an in vivo subject, and the method is a treatmentfor hearing loss and/or vestibular dysfunction (e.g., wherein thegeneration of inner ear hair cells from the expanded population of stemcells results in partial or full recovery of hearing loss and/orimproved vestibular function).

28. The method of any of the preceding embodiments, wherein the stemcell population is of an in vivo subject, and wherein the method furthercomprises delivering a drug to the subject (e.g., for treatment of adisease and/or disorder unrelated to hearing loss and/or vestibulardysfunction) at a higher concentration than a known safe maximum dosageof the drug for the subject (e.g., the known safe maximum dosage ifdelivered in the absence of the generation of inner ear hair cellsresulting from the method) (e.g., due to a reduction or elimination of adose-limiting ototoxicity of the drug).

29. The method of any of the preceding embodiments, further comprisingperforming high throughput screening using the generated inner ear haircells.

30. The method of embodiment 29, comprising using the generated innerear hair cells to screen molecules for toxicity against inner ear haircells.

31. The method of embodiment 29, comprising using the generated innerear hair cells to screen molecules for ability to improve survival ofinner ear hair cells (e.g., inner ear hair cells exposed to saidmolecules).

32. A method of producing an expanded population of stem cells, themethod comprising:

-   -   administering or causing to be administered to a stem cell        population (e.g., of an in vitro, ex vivo, or in vivo        sample/subject) both of (i) and (ii):

(i) a GSK3-beta inhibitor and/or Wnt agonist, and

(ii) a notch agonist and/or HDAC inhibitor,

thereby proliferating stem cells in the stem cell population andresulting in an expanded population of stem cells.

33. The method of embodiment 32, wherein the stem cell populationcomprises Lgr5+ cells.

34. The method of embodiment 33, wherein the stem cell populationcomprises post-natal stem cells.

35. The method of any of embodiments 32 to 34, wherein the stem cellpopulation comprises epithelial stem cells.

36. The method of any of embodiments 32 to 35, wherein the administeringstep comprises administering or causing to be administered to the stemcell population a notch agonist that is also an HDAC inhibitor.

37. The method of any of embodiments 32 to 36, wherein the administeringstep comprises administering or causing to be administered to the stemcell population a notch agonist that comprises a synthetic molecule.

38. The method of any of embodiments 32 to 37, wherein the administeringstep comprises administering or causing to be administered to the stemcell population VPA (e.g., in a pharmaceutically acceptable form (e.g.,salt)) (e.g., where VPA is a notch agonist that is also an HDACinhibitor).

39. The method of any of embodiments 32 to 38, wherein the administeringstep comprises administering or causing to be administered to the stemcell population a Wnt agonist that is also a GSK3-beta inhibitor.

40. The method of any of embodiments 32 to 39, wherein the administeringstep comprises administering or causing to be administered to the stemcell population a Wnt agonist comprising a synthetic molecule.

41. The method of any of embodiments 32 to 40, wherein the administeringstep comprises administering or causing to be administered to the stemcell population CHIR99021 (e.g., in a pharmaceutically acceptable form(e.g., salt)) (e.g., where CHIR99021 is a GSK3 beta inhibitor).

42. The method of any of embodiments 32 to 41, wherein the administeringstep is carried out by performing one or more injections into the ear(e.g., transtympanically into the middle ear and/or inner ear).

43. The method of any of embodiments 32 to 42, wherein the administeringstep comprises administering the notch agonist and/or HDAC inhibitor ina pulsatile manner and administering the GSK3-beta inhibitor and/or Wntagonist in a sustained manner.

44. The method of any of embodiments 32 to 43, wherein the stem cellsare inner ear stem cells.

45. The method of any of embodiments 32 to 44, further comprisingperforming high throughput screening using the generated expandedpopulation of stem cells.

46. The method of embodiment 45, comprising using the generated stemcells to screen molecules for toxicity against stem cells and/or theirprogeny.

47. The method of embodiment 45, comprising using the generated stemcells to screen molecules for ability to improve survival of stem cellsand/or their progeny.

48. A method of treating a subject who has, or is at risk of developing,hearing loss and/or vestibular dysfunction, the method comprising:

identifying a subject who has experienced, or is at risk for developing,hearing loss and/or vestibular dysfunction,

administering or causing to be administered to the subject both of (i)and (ii):

-   -   (i) a GSK3-beta inhibitor and/or Wnt agonist, and    -   (ii) a notch agonist and/or HDAC inhibitor,

thereby treating or preventing the hearing loss and/or vestibulardysfunction in the subject.

49. The method of embodiment 48, wherein the stem cell populationcomprises Lgr5+ cells.

50. The method of embodiment 48 or 49, wherein the stem cell populationcomprises post-natal cells.

51. The method of any of embodiments 48 to 50, wherein the stem cellpopulation comprises epithelial stem cells.

52. The method of any of embodiments 48 to 51, wherein the administeringstep comprises administering or causing to be administered to thesubject a notch agonist that is also an HDAC inhibitor.

53. The method of any of embodiments 48 to 52, wherein the administeringstep comprises administering or causing to be administered to thesubject a notch agonist comprising a synthetic molecule.

54. The method of any of embodiments 48 to 53, wherein the administeringstep comprises administering or causing to be administered to thesubject VPA (e.g., in a pharmaceutically acceptable form (e.g., salt))(e.g., where VPA is a notch agonist that is also an HDAC inhibitor).

55. The method of any of embodiments 48 to 54, wherein the administeringstep comprises administering or causing to be administered to thesubject a Wnt agonist that is also a GSK3-beta inhibitor.

56. The method of any of embodiments 48 to 55, wherein the administeringstep comprises administering or causing to be administered to thesubject a Wnt agonist comprising a synthetic molecule.

57. The method of any of embodiments 48 to 56, wherein the administeringstep comprises administering or causing to be administered to thesubject CHIR99021 (e.g., in a pharmaceutically acceptable form (e.g.,salt)) (e.g., where CHIR99021 is a GSK3-beta inhibitor).

58. The method of any of embodiments 48 to 57, wherein the step ofadministering is carried out by performing one or more injections intothe ear (e.g., transtympanically into the middle ear and/or inner ear).

59. The method of any of embodiments 48 to 58, comprising administeringthe notch agonist and/or the HDAC inhibitor in a pulsatile manner andadministering the GSK3-beta inhibitor and/or Wnt agonist in a sustainedmanner.

60. A composition comprising the first or second composition of any ofthe preceding embodiments.

61. A composition comprising (a) (i) a GSK3-beta inhibitor or derivativeor a pharmaceutically acceptable salt thereof and/or Wnt agonist orderivative or a pharmaceutically acceptable salt thereof, and (ii) anotch agonist or derivative or a pharmaceutically acceptable saltthereof and/or HDAC inhibitor or derivative or a pharmaceuticallyacceptable salt thereof, and (b) a pharmaceutically acceptable carrieror excipient.

62. A composition comprising (a) (i) a GSK3-beta inhibitor or derivativeor a pharmaceutically acceptable salt thereof, and (ii) a notch agonistor derivative or a pharmaceutically acceptable salt thereof and (b) apharmaceutically acceptable carrier or excipient.

63. A composition comprising (a) (i) a Wnt agonist or derivative or apharmaceutically acceptable salt thereof, and (ii) a notch agonist orderivative or a pharmaceutically acceptable salt thereof; and (b) apharmaceutically acceptable carrier or excipient.

64. A composition comprising (a) (i) a GSK3-beta inhibitor or derivativeor a pharmaceutically acceptable salt thereof and (ii) a HDAC inhibitoror derivative or a pharmaceutically acceptable salt thereof, and (b) apharmaceutically acceptable carrier or excipient.

65. A composition comprising (a) (i) a Wnt agonist or derivative or apharmaceutically acceptable salt thereof, and (ii) a HDAC inhibitor orderivative or a pharmaceutically acceptable salt thereof, and (b) apharmaceutically acceptable carrier or excipient.

65A. The composition of any of embodiments 61 to 65 wherein thecomposition comprises a GSK3-beta inhibitor.

65B. The composition of any of embodiments 61 to 65 wherein thecomposition comprises a Wnt agonist.

65C. The composition of any of embodiments 61 to 65 wherein thecomposition comprises a notch agonist.

65D. The composition of any of embodiments 61 to 65 wherein thecomposition comprises a HDAC inhibitor,

66. A pharmaceutical composition comprising a GSK3-beta inhibitor and anotch agonist in lyophilized form.

67. A pharmaceutical composition comprising a GSK3-beta inhibitor and anotch agonist in hydrated form.

68. The pharmaceutical composition of embodiment 66 or 67, wherein theGSK3-beta inhibitor is CHIR99021 (e.g., in a pharmaceutically acceptableform (e.g., salt)).

69. The pharmaceutical composition of any of embodiments 66 to 68,wherein the notch agonist is VPA (e.g., in a pharmaceutically acceptableform (e.g., salt)).

70. A method of generating inner ear hair cells, the method comprising:

-   -   proliferating stem cells in an initial stem cell population        (e.g., of an in vitro, ex vivo, or in vivo sample/subject),        resulting in an expanded population of stem cells (e.g., such        that the expanded population is at least 2 times, 3 times, 5        times, 10 times, or 20 times greater than the initial stem cell        population); and    -   exposing the expanded population of stem cells to a GSK3-beta        inhibitor and/or a Wnt agonist, and, optionally, a notch        inhibitor, thereby facilitating generation of inner ear hair        cells from the expanded population of stem cells.

71. A method of generating inner ear hair cells, the method comprisingadministering CHIR99021 (e.g., in a pharmaceutically acceptable form(e.g., salt)) to a cell population in an inner ear of a subject, therebyfacilitating generation of inner ear hair cells.

72. A method of generating inner ear hair cells, the method comprisingproliferating post-natal LGR5+ cells in an initial population (e.g., ofan in vitro, ex vivo, or in vivo sample/subject), resulting in anexpanded population of LGR5+ cells (e.g., such that the expandedpopulation is at least 2 times, 3 times, 5 times, 10 times, or 20 timesgreater than the initial stem cell population), said expanded populationof LGR5+ cells resulting in generation of inner ear hair cells.

73. A method of treating a disease or disorder, the method comprisingproliferating post-natal Lgr5+ epithelial cells in an initial populationof a subject (in vivo), resulting in an expanded population of Lgr5+epithelial cells (e.g., such that the expanded population is at least 2times, 3 times, 5 times, 10 times, or 20 times greater than the initialpost-natal Lgr5+ epithelial cell population).

74. A method of proliferating stem cells, the method comprising:

-   -   administering or causing to be administered to a stem cell        population (e.g., of an in vitro, ex vivo, or in vivo        sample/subject) both of (i) and (ii):        -   (i) a GSK3-beta inhibitor and/or Wnt agonist, and        -   (ii) a notch agonist and/or HDAC inhibitor,

thereby proliferating stem cells in the stem cell population andresulting in an expanded population of stem cells; and

-   -   generating inner ear hair cells from the expanded population of        stem cells.

75. A kit comprising:

(a) a set of one or more compositions, the set comprising (i) and (ii):

-   -   (i) a GSK3-beta inhibitor and/or Wnt agonist, and    -   (ii) a notch agonist and/or HDAC inhibitor,

each of the one or more compositions provided in a pharmaceuticallyacceptable carrier and

(b) instructions for using the set of one or more compositions to treatan inner ear disorder.

76. The kit of embodiment 75, wherein the set of one or morecompositions also comprises a TGFβ inhibitor.

77. The kit of embodiment 75 or 76, wherein the set of one or morecompositions also comprises an ROS inhibitor.

78. The kit of embodiment 77, wherein the ROS inhibitor is vitamin C ora derivative thereof.

79. The kit of any of embodiments 75 to 77, wherein the set of one ormore compositions is/are in a form that can be injected (e.g. viasyringe).

80. The kit of embodiment 79, wherein the set of one or morecompositions is/are in a form that can be injected into the middle ear.

81. A method for enhancing the stem cell potential of a population ofcochlear supporting cells.

82. The method of embodiment 81 wherein the method comprises activatingthe Wnt pathway in the cochlear supporting cell population.

83. The method of any preceding embodiment wherein the method comprisesactivating the Wnt pathway in the cochlear supporting cell populationupstream of c-myc.

84. The method of any preceding enumerated embodiment wherein thecochlear supporting cells are endogenous to the Organ of Corti.

85. The method of any preceding embodiment wherein the cochlearsupporting cell population comprises a population of Lgr5⁺ supportingcells.

86. The method of any preceding embodiment wherein the cochlearsupporting cell population comprises Lgr5⁺ supporting cells and themethod further comprises inducing the Lgr5⁺ supporting cells divide,giving rise to a multi-potent Lgr5⁺ daughter cells.

87. The method of any preceding embodiment in which the parent and/ordaughter Lgr5⁺ cells subsequently differentiate into hair cell(s).

88. The method of any preceding embodiment in which Lgr5 expression in agiven supporting cell is maintained within 25% of its baseline level.

89. A method of any preceding embodiment in which Lgr5 is upregulated ina supporting cell by a factor of at least 1.25, 1.5, 2, 5, 10, 100, or1,000 of its baseline expression level.

90. A method of any preceding embodiment in which Lgr5 is upregulated ina supporting cell population, on average, by a factor of at least 1.25,1.5, 2, 5, 10, 100, or 1,000 of the baseline expression level, for thepopulation, and the supporting cell subsequently proliferates.

91. A method of any preceding embodiment in which Lgr5 is upregulated ina supporting cell population, on average, by a factor of at least 1.25,1.5, 2, 5, 10, 100, or 1,000 of the baseline expression level, for thepopulation, and members of the supporting cell population divide anddifferentiate into a hair cell

92. A method for inducing the self-renewal of stem/progenitor supportingcells comprised by a cochlear cell population, the method comprisinginducing the stem/progenitor cells to proliferate while maintaining, inthe daughter cells, the capacity to differentiate into hair cells.

93. The method of embodiment 92 wherein the daughter cells are permittedto differentiate into hair cells.

94. The method of embodiment 92 wherein the daughter cells are inducedto differentiate into hair cells.

95. The method of embodiment 92 wherein the daughter cells are inducedto differentiate into hair cells by activating the Wnt pathway upstreamof the c-myc gene and without any genetic modification of thepopulation.

96. The method of embodiment 92 wherein the daughter cells are inducedto differentiate into hair cells by activating the Wnt pathway upstreamof the c-myc gene with small organic molecules that transiently inducesuch activity.

97. The of any of embodiments 92-96 wherein the supporting cellpopulation includes LGR5⁺ supporting cells that are endogenous to theOrgan of Corti.

98. A composition having the capacity to induce self-renewal of apopulation of supporting cells by activation or inhibition of a pathwayinvolved in inducing stem cell properties.

99. The composition of embodiment 98 wherein the pathway is selectedfrom the group consisting of Wnt, HDAC, TGF-beta, RAR, DKK1, andcombinations thereof.

100. The composition of embodiment 98 or 99 wherein the compositioncomprises a small organic molecule that activates or inhibits thepathway.

101. The composition of embodiment 98, 99 or 100 wherein the compositioncomprises a Biocompatible Matrix.

102. The composition of any of embodiments 98-101 wherein when appliedin vitro to a supporting cell population, the composition induces thepopulation to proliferate to a high degree and in high purity in a StemCell Proliferation Assay, and also allows the population todifferentiate into a high purity population of hair cells in a Stem CellDifferentiation Assay.

103. The composition of any of embodiments 98-102 wherein thecomposition induces and maintains stem cell properties by proliferatingthe supporting cell population to produce stem cells that can divide formany generations and maintain the ability to have a high proportion ofthe resulting cells differentiate into hair cells.

104. The composition of any of embodiments 98-102 wherein theproliferating cells express stem cell markers including one or more ofLgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3,Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31,Utf1, Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3,Smad1, Smad2, smad2/3, smad4, smad5, and smad7.

105. A method for increasing the cell density of supporting cells in apopulation of cochlear cells comprising activating pathways andmechanisms that induce stem cell properties in the supporting cells,proliferating the activated supporting cells while maintaining themulti-potent character of the supporting cells in the newly formeddaughter cells and thereafter allowing or inducing the expandedpopulation to differentiate into hair cells to form an expanded cochlearcell population wherein the cell density of hair cells in the expandedcochlear cell population exceeds the cell density of hair cells in theoriginal (non-expanded) cochlear cell population.

106. The method of embodiment 105 wherein the expanded population isallowed differentiate into hair cells.

107. The method of embodiment 105 wherein the expanded population isinduced to differentiate into hair cells.

108. The method of any of embodiments 105-107 wherein the supportingcell population is comprised by a cochlear tissue.

109. The method of any of embodiments 105-108 wherein the supportingcell population is an in vitro supporting cell population.

110. The method of any of embodiments 105-108 wherein the supportingcell population is an in vivo supporting cell population.

111. The method of any of embodiments 105-110 wherein the proliferationstage is preferably controlled to substantially maintain the nativeorganization of the cochlear structure.

112. The method of any of embodiments 105-111 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc andwithout any genetic modification of the population.

113. The method of any of embodiments 105-112 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc witha small organic molecule and without any genetic modification of thepopulation.

114. The method of any of embodiments 105-113 wherein the supportingcell population includes supporting cells that are Lgr5⁺ and endogenousto the Organ of Corti.

115. A method for increasing the cell density of Lgr5⁺ supporting cellsin a population of cochlear cells, the method comprising activatingpathways and mechanisms that induce or maintain stem cell properties inthe Lgr5⁺ supporting cells, proliferating the activated Lgr5⁺ supportingcells while maintaining such stem cell properties and thereafterallowing or inducing the expanded population to differentiate into haircells to form an expanded cochlear cell population wherein the celldensity of hair cells in the expanded cochlear cell population exceedsthe cell density of hair cells in the original (non-expanded) cochlearcell population.

116. The method of embodiment 115 wherein the expanded population isinduced to differentiate into hair cells.

117. The method of embodiment 115 wherein the expanded population isinduced to differentiate into hair cells.

118. The method of any of embodiments 115-117 wherein the supportingcell population is comprised by a cochlear tissue.

119. The method of any of embodiments 115-118 wherein the supportingcell population is an in vitro supporting cell population.

120. The method of any of embodiments 115-118 wherein the supportingcell population is an in vivo supporting cell population.

121. The method of any of embodiments 115-120 wherein the proliferationstage is preferably controlled to substantially maintain the nativeorganization of the cochlear structure.

122. The method of any of embodiments 115-121 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc andwithout any genetic modification of the population.

123. The method of any of embodiments 115-122 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc witha small organic molecule and without any genetic modification of thepopulation.

124. The method of any of embodiments 115-123 wherein the supportingcell population includes Lgr5⁺ cells endogenous to the Organ of Corti.

125. A method for increasing the cell density of hair cells in aninitial population of cochlear cells, the initial population compriseshair cells, Lgr⁻ supporting cells, and Lgr5⁺ supporting cells, themethod comprising administering to the initial population a Stem CellProliferator composition that contains a Stemness Driver and aDifferentiation Inhibitor wherein the composition has the capacity toinduce the expansion of the number of Lgr5⁺ supporting cells in thepopulation in a Stem Cell Proliferation Assay, and allows Lgr5⁺supporting cells within the population to differentiate into apopulation of hair cells in a Stem Cell Differentiation Assay.

126. The method of embodiment 125 wherein the expanded population isinduced to differentiate into hair cells.

127. The method of embodiment 125 wherein the expanded population isinduced to differentiate into hair cells.

128. The method of any of embodiments 125-127 wherein the supportingcell population is comprised by a cochlear tissue.

129. The method of any of embodiments 125-128 wherein the supportingcell population is an in vitro supporting cell population.

130. The method of any of embodiments 125-128 wherein the supportingcell population is an in vivo supporting cell population.

131. The method of any of embodiments 125-130 wherein the proliferationstage is preferably controlled to substantially maintain the nativeorganization of the cochlear structure.

132. The method of any of embodiments 125-131 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc andwithout any genetic modification of the population.

133. The method of any of embodiments 125-132 wherein the proliferationis transiently induced by induction of a pathway upstream of c-myc witha small organic molecule and without any genetic modification of thepopulation.

134. The method of any of embodiments 125-133 wherein the supportingcell population includes Lgr5⁺ cells endogenous to the Organ of Corti.

135. The method of any of embodiments 125-134 wherein the methodproduces stem cells in a Stem Cell Proliferation Assay that express oneor more stem cells markers selected from the group consisting of Sox2,Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3,Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tcl1,Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1,Smad2, smad2/3, smad4, smad5, and smad7.

136. The method of any of embodiments 125-135 wherein the fraction ofcells in the population that are Lgr5⁺ is increased.

137. A method for increasing the cell density of hair cells in aninitial population of cochlear cells comprising hair cells andsupporting cells, the method comprising

selectively expanding the number of supporting cells in the initialpopulation to form an intermediate cochlear cell population wherein theratio of the number of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of the number ofsupporting cells to hair cells in the initial cochlear cell population,and

selectively expanding the number of hair cells in the intermediatecochlear cell population to form an expanded cochlear cell populationwherein the ratio of the number of hair cells to supporting cells in theexpanded cochlear cell population exceeds the ratio of the number ofhair cells to supporting cells in the intermediate cochlear cellpopulation.

138. The method of embodiment 137 wherein the intermediate population isinduced to differentiate into hair cells.

139. The method of embodiment 137 wherein the intermediate population isinduced to differentiate into hair cells.

140. The method of any of embodiments 137-139 wherein the initialcochlear cell population is comprised by a cochlear tissue.

141. The method of any of embodiments 137-139 wherein the initialcochlear cell population is an in vitro population.

142. The method of any of embodiments 137-139 wherein the initialcochlear cell population is an in vivo population.

143. The method of any of embodiments 137-142 wherein the selectiveexpansion of the supporting cells is controlled to substantiallymaintain the native organization of the cochlear structure.

144. The method of any of embodiments 137-143 wherein the selectiveexpansion of the supporting cells is transiently induced by induction ofa pathway upstream of c-myc and without any genetic modification of thepopulation.

145. The method of any of embodiments 137-144 wherein the selectiveexpansion of the supporting cells is induced by induction of a pathwayupstream of c-myc with a small organic molecule and without any geneticmodification of the population.

146. The method of any of embodiments 137-145 wherein the supportingcell population includes Lgr5⁺ cells endogenous to the Organ of Corti.

147. The method of any of embodiments 137-146 wherein the methodproduces stem cells in a Stem Cell Proliferation Assay that express oneor more stem cells markers selected from the group consisting of Lgr5,Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3,Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1,Tcl1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3,Smad1, Smad2, smad2/3, smad4, smad5, and smad7.

148. The method of any of embodiments 137-147 wherein the fraction ofcells in the intermediate population that are Lgr5⁺ is greater than thefraction of cells in the initial population that are Lgr5⁺.

149. A method for increasing the number of Lgr5⁺ supporting cells orincreasing the Lgr5 activity in an initial population of cochlear cells,wherein the initial population comprises supporting cells and haircells.

150. The method of embodiment 149 wherein an intermediate population isformed in which the number of Lgr5⁺ supporting cells is expandedrelative to the initial population.

151. The method of embodiment 149 or 150 wherein an intermediatepopulation is formed in which the Lgr5⁺ activity of the supporting cellsrelative to the initial population is increased.

152. The method of any of embodiments 149-151 wherein the number ofLgr5⁺ cells is increased relative to the initial cell population byactivating Lgr5⁺ expression in cell types that normally lack or havevery low levels of Lgr5.

153. The method of any of embodiments 149-151 wherein an intermediatepopulation is formed in which the number of Lgr5⁺ supporting cells isexpanded and the Lgr5 activity is increased relative to the initialcochlear cell population.

154. The method of any of embodiments 149-151 wherein an intermediatepopulation is formed in which the number of Lgr5⁺ supporting cells isexpanded and the Lgr5 activity is increased relative to the initialcochlear cell population and the number of hair cells in theintermediate cochlear cell population is thereafter selectively expandedto form an expanded cochlear cell population wherein the ratio of haircells to supporting cells in the expanded cochlear cell populationexceeds the ratio of the number of hair cells to supporting cells in theintermediate cochlear cell population.

155. The method of any of embodiments 81-97 and 105-154 wherein themethod produces a population of Lgr5⁺ cells that are in S-phase.

156. The method of any of embodiments 81-97 and 105-154 wherein themethod is used with adult mammal cochlear cells and the method producesa population of adult mammalian Lgr5⁺ cells that are in S-phase.

157. The method of any of embodiments 81-97 and 105-156 wherein themethod comprises contacting the cochlear cell population with a StemCell Proliferator.

158. The method of any of embodiments 81-97 and 105-156 wherein themethod comprises contacting the cochlear cell population with a StemCell Proliferator containing a Stemness Driver.

159. The method of any of embodiments 81-97 and 105-156 wherein themethod comprises contacting the cochlear cell population with aDifferentiation Inhibitor.

160. The method of any of embodiments 81-97 and 105-156 wherein themethod comprises contacting the cochlear cell population with a StemCell Proliferator containing a Stemness Driver and a DifferentiationInhibitor.

161. The method of any of embodiments 81-97 and 105-160 wherein aStemness Driver is used to drive the proliferation of Lgr5⁺ stem cells.

162. The method of any of embodiments 81-97 and 105-161 wherein aStemness Driver is used to induce differentiation of LGR5+ cells to haircells when a Differentiation Inhibitor is not present at an EffectiveDifferentiation Inhibition Concentration.

163. The method of any of embodiments 81-97 and 105-162 wherein theStemness Driver that drives both proliferation and differentiationcomprises GSK3Beta inhibitors and Wnt agonists.

164. The method of any of embodiments 81-97 and 105-163 wherein thecomposition comprises a Stemness Driver and a Differentiation Inhibitorin a formulation that releases the Stemness Driver and Differentiationinhibitor at different rates in a Release Assay.

165. The method of any of embodiments 81-97 and 105-163 wherein thecomposition comprises a Stemness Driver and a Differentiation Inhibitorin a formulation that releases the Stemness Driver and Differentiationinhibitor at a constant, sustained, extended, delayed or pulsatile rateof release of the active agent into the inner ear environment.

166. The method or composition of any of the previous embodiments inwhich the Differentiation Inhibitor may be a Notch agonist or HDACinhibitor.

167. The method or composition of any of the previous embodiments inwhich there is a first Proliferation Period with an Effective StemnessDriver Concentration and an Effective Differentiation InhibitionConcentration of a Differentiation Inhibitor, followed by aDifferentiation Period with an Effective Stemness Driver Concentrationand without an Effective Differentiation Inhibition Concentration of aDifferentiation Inhibitor.

168. The method or composition of any of the previous embodiments inwhich the Stemness Driver and Differentiation Inhibitor are provided tothe cochlear cells in a formulation that releases the Stemness Driverand Differentiation inhibitor at different rates.

169. The method or composition of any of the previous embodiments inwhich the formulation provides a constant, sustained, extended, delayedor pulsatile rate of release of the Stemness Driver and theDifferentiation Inhibitor into the inner ear environment.

170. The method or composition of any of the previous embodiments inwhich the formulation releases the Stemness Driver and DifferentiationInhibitor in a manner to provide a first Proliferation Period with anEffective Stemness Driver Concentration and an Effective DifferentiationInhibition Concentration of the Differentiation Inhibitor, followed by aDifferentiation Period with an Effective Stemness Driver Concentrationand without an Effective Differentiation Inhibition Concentration of theDifferentiation Inhibitor.

171. The method or composition of any of the previous embodiments inwhich there is a first Proliferation Period with an Effective StemnessDriver Concentration of a Wnt agonist or GSK3Beta inhibitor and anEffective Differentiation Inhibition Concentration of a Notch agonist orHDAC inhibitor, followed by a Differentiation Period with an EffectiveStemness Driver Concentration of a Wnt agonist or GSK3Beta inhibitor andwithout an Effective Differentiation Inhibition Concentration of a Notchagonist or HDAC inhibitor.

172. The method or composition of embodiment 171 wherein the formulationprovides a constant, sustained, extended, delayed or pulsatile rate ofrelease of the Wnt agonist or GSK3Beta inhibitor and the Notch agonistor HDAC inhibitor into the inner ear environment.

173. The method or composition of embodiment 171 or 172 wherein theformulation releases the Wnt agonist or GSK3Beta inhibitor and the Notchagonist or HDAC inhibitor in a manner to provide a first ProliferationPeriod with an Effective Stemness Driver Concentration of the Wntagonist or GSK3Beta inhibitor and an Effective DifferentiationInhibition Concentration of the Notch agonist or HDAC inhibitor,followed by a Differentiation Period with an Effective Stemness DriverConcentration of the Wnt agonist or GSK3Beta inhibitor and without anEffective Differentiation Inhibition Concentration of the Notch agonistor HDAC inhibitor.

174. The method or composition of any of the previous embodimentswherein the Differentiation inhibitor is also a Stemness Driver.

175. The method or composition of any of the previous embodimentswherein the Differentiation inhibitor is a Notch agonist and is also aStemness Driver.

176. The method or composition of any of the previous embodimentswherein the Differentiation inhibitor is Valproic Acid.

177. The method or composition of any of the previous embodimentswherein if the Differentiation Inhibitor is also a Stemness Driver, theconcentration of the Differentiation Inhibitor falls below the EffectiveDifferentiation Inhibition Concentration during the DifferentiationPeriod.

178. The method or composition of any of the previous embodimentswherein the Differentiation inhibitor and Stemness Driver are becontained within a sustained release polymer gel.

179. The method or composition of any of the previous embodimentswherein the gel is (or is adapted to be) injected through a needle andbecome solid in the middle ear space.

180. The method or composition of embodiment 179 wherein the gelcomprises a thermoreversible polymer.

181. The method or composition of embodiment 179 wherein the gelcomprises Poloxamer 407.

182. The method or composition of any previous embodiment wherein theNotch activity level in supporting cells remains at least 10, 20, 30,40, 50, 60, 70, 80, 90, or 100% of the Notch activity level insupporting cells in the native state.

183. A method of and compositions for generating hair cells, the methodcomprising: administering or causing to be administered to a stem cellpopulation (e.g., of an in vitro, ex vivo, or in vivo sample/subject) acomposition comprising both of (i) and (ii): (i) a GSK3-beta inhibitor(or a derivative or pharmaceutically acceptable salt thereof) and/or Wntagonist (or a derivative or pharmaceutically acceptable salt thereof),and (ii) a Notch agonist (or a derivative or pharmaceutically acceptablesalt thereof) and/or HDAC inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof), thereby proliferating stem cells in the stemcell population and resulting in an expanded population of stem cells;and exposing the expanded population of stem cells to a GSK3-betainhibitor (or a derivative or pharmaceutically acceptable salt thereof)and/or a Wnt agonist (or a derivative or pharmaceutically acceptablesalt thereof), and, optionally, a notch inhibitor (or a derivative orpharmaceutically acceptable salt thereof), thereby facilitatinggeneration of inner ear hair cells from the expanded population of stemcells.

184. A method for preventing or treating auditory impairments in asubject comprising administering to said subject an effective amount ofa composition comprising (a) (i) an HDAC inhibitor and/or Notchactivator and (ii) a GSK3-beta inhibitor, a derivative thereof [e.g., aderivative of an HDAC inhibitor, a derivative of a Notch activator,and/or a derivative of a GSK3-beta inhibitor], a pharmaceuticallyacceptable salt thereof [e.g., a pharmaceutically acceptable salt of anHDAC inhibitor, a pharmaceutically acceptable salt of a Notch activator,and/or a pharmaceutically acceptable salt of a GSK3-beta inhibitor], ora combination thereof and (b) a pharmaceutically acceptable carrier orexcipient, so as to treat auditory impairments in the subject. Thus, forexample, the composition may comprise (a) an HDAC inhibitor (or aderivative or pharmaceutically acceptable salt thereof) and a GSK3-betainhibitor (or a derivative or pharmaceutically acceptable salt thereof),and (b) a pharmaceutically acceptable carrier or excipient. By way offurther example, the composition may comprise (a) a Notch activator (ora derivative or pharmaceutically acceptable salt thereof) and aGSK3-beta inhibitor (or a derivative or pharmaceutically acceptable saltthereof), and (b) a pharmaceutically acceptable carrier or excipient. Byway of further example, the composition may comprise (a) an HDACinhibitor (or a derivative or pharmaceutically acceptable salt thereof),a Notch activator (or a derivative or pharmaceutically acceptable saltthereof), and a GSK3-beta inhibitor (or a derivative or pharmaceuticallyacceptable salt thereof), and (b) a pharmaceutically acceptable carrieror excipient.

185. A method for identifying agents that proliferate hair cellprogenitors and/or increase numbers of hair cells, and also agents thatprotect supporting cells and/or hair cells (e.g. to support theirsurvival), and also for identifying agents that are toxic or not toxicto supporting cells or differentiated progeny including hair cells.

186. A method for preventing or treating auditory impairments in asubject in need of treatment comprising administering to said subject aneffective amount of a composition comprising, an HDAC inhibitor and/orNotch activator and a GSK3beta inhibitor or derivative thereof orpharmaceutically acceptable salt thereof and an acceptable carrier orexcipient, so as to treat auditory impairments in the subject.

187. A method for inhibiting the loss or death of the cells of theauditory system in a subject comprising administering to said subject aneffective amount of a composition described herein or derivative thereofor pharmaceutically acceptable salt thereof and an acceptable carrier orexcipient, thereby inhibiting loss or death of the cells of the auditorysystem in the subject.

188. A method for maintaining or promoting the growth of cells of theauditory system in a subject comprising administering to said subject acomposition comprising as an agent described herein or derivativethereof or pharmaceutically acceptable salt thereof and an acceptablecarrier or excipient in an effective amount so as to augment or initiateendogenous repair, thereby maintaining or promoting the growth of cellsof the auditory system in the subject.

189. The method of any of the previous embodiments wherein the averagein vitro Lgr5 Activity in the cell population upon completion of theStem Cell Proliferation Assay is no less than 25, 50, 75, or 90% of theaverage in vitro Lgr5 Activity in the cell population at the start ofthe Stem Cell Proliferation Assay.

190. The method of any of the previous embodiments wherein the averagein vitro notch activity in the cell population upon completion of theStem Cell Proliferation Assay is no less than 25, 50, 75, or 90% of theaverage in vitro notch activity in the cell population used at the startof the Stem Cell Proliferation Assay.

191. A method for expanding a population of cochlear stem cells in acochlear tissue comprising a parent population of cells, the parentpopulation including supporting cells, the method comprising contactingthe cochlear tissue with a Stem Cell Proliferator to form an expandedpopulation of cells in the cochlear tissue, the Stem Cell Proliferatoris capable (i) in a Stem Cell Proliferation Assay of increasing thenumber of Lgr5⁺ cells in a Stem Cell Proliferation Assay cell populationby a factor of at least 10 and (ii) in a Stem Cell Differentiation Assayof forming hair cells from a cell population comprising Lgr5⁺ cells.

192. The method of embodiment 191, wherein the at least one Stem CellProliferator is capable, in a Stem Cell Proliferation Assay, ofincreasing the number of Lgr5⁺ cells in the Stem Cell ProliferationAssay cell population by a factor of at least 50.

193. The method of embodiment 191, wherein the Stem Cell Proliferator atleast one Stem Cell Proliferator is capable, in the Stem CellProliferation Assay, of increasing the number of Lgr5⁺ cells in a StemCell Proliferation Assay cell population by a factor of at least 100.

194. The method as in any of embodiments 191-193 wherein the expandedpopulation of cells comprises a greater number of hair cells than theparent population.

195. The method as in any of embodiments 191-194 wherein the fraction oftotal cells in the Stem Cell Proliferation Assay cell population thatare Lgr5⁺ cells increases from the start to the end of the Stem CellProliferation assay by at least 2-fold.

196. The method as in any of embodiments 191-195 wherein the fraction oftotal cells in the Stem Cell Differentiation Assay cell population thatare hair cells increases from the start to the end of the Stem CellDifferentiation assay by at least 2-fold.

197. The method as in any of embodiments 191-196 wherein the fraction oftotal cells in the Stem Cell Proliferation Assay cell population thatare hair cells decreases from the start to the end of the Stem CellProliferation assay by at least 25%.

198. The method as in any of embodiments 191-197 wherein the averageLgr5⁺ activity per cell in the Stem Cell Proliferation Assay cellpopulation increases in the Stem Cell Proliferation Assay by at least10%.

199. The method as in any of embodiments 191-198 wherein the average invitro Lgr5 Activity in the cell population upon completion of the StemCell Proliferation Assay is no less than 25, 50, 75, or 90% of theaverage in vitro Lgr5 Activity in the cell population at the start ofthe Stem Cell Proliferation Assay.

200. The method as in any of embodiments 191-199 wherein the average invitro notch activity in the cell population upon completion of the StemCell Proliferation Assay is no less than 25, 50, 75, or 90% of theaverage in vitro notch activity in the cell population used at the startof the Stem Cell Proliferation Assay.

201. The method as in any of embodiments 191-200 wherein the cochleartissue maintains Native Morphology.

202. The method as in any of embodiments 191-201 wherein the at leastone Stem Cell Proliferator is dispersed in a Biocompatible Matrix.

203. The method of embodiment 202 wherein the Biocompatible Matrix is aBiocompatible Gel or Foam.

204. The method as in any of embodiments 191-203 wherein the compositionis a controlled release formulation.

205. The method as in any of embodiments 191-204 wherein the cochleartissue is an in vivo cochlear tissue.

206. The method as in any of embodiments 191-204 wherein the cochleartissue is an ex vivo cochlear tissue.

207. The method as in any of embodiments 191-205 wherein the methodproduces a population of Lgr5⁺ cells that are in S-phase.

208. The method as in any of embodiments 191-207 wherein the at leastone Stem Cell Proliferator comprises both a Stemness Driver and aDifferentiation Inhibitor.

209. The method as in any of embodiments 191-208 wherein the contactingprovides to the cochlear tissue:

in an Initial Phase, at least an Stemness Driver Concentration and atleast an Effective Differentiation Inhibition Concentration of theDifferentiation Inhibitor; and

in a Subsequent Phase, at least an Stemness Driver Concentration andless than an Effective Differentiation Inhibition Concentration of theDifferentiation Inhibitor.

210. The method as in any of embodiments 191-209, wherein the cochleartissue is in a subject, and wherein the contacting the cochlear tissuewith the composition is achieved by administering the compositiontrans-tympanically to the subject.

211. The method of embodiment 210 wherein the contacting the cochleartissue with the composition results in improved Auditory Functioning ofthe subject.

212. A composition comprising a Biocompatible Matrix and at least oneStem Cell Proliferator, wherein the at least one Stem Cell Proliferatoris capable, in a Stem Cell Proliferation Assay, of expanding an initialtest population of LGR5⁺ cells to create an expanded test population,and wherein the expanded test population has at least 10-fold more LGR5⁺cells than does the initial test population.

213. The composition of embodiment 212 wherein the at least one StemCell Proliferator is capable, in the Stem Cell Proliferation Assay, ofincreasing the number of Lgr5⁺ cells in a Stem Cell Proliferation Assaycell population by a factor of at least 50.

214. The composition of embodiment 212, wherein the at least one StemCell Proliferator is capable, in the Stem Cell Proliferation Assay, ofincreasing the number of Lgr5+ cells in a Stem Cell Proliferation Assaycell population by a factor of at least 100.

215. The composition of any of embodiments 212-214, wherein the at leastone Stem Cell Proliferator is dispersed in a Biocompatible Matrix.

216. The composition of embodiment 215, wherein the Biocompatible Matrixis a Biocompatible Gel or Foam.

217. The composition of embodiment 215 wherein the at least one StemCell Proliferator is capable, in the Stem Cell Proliferation Assay, ofincreasing the average Lgr5+ activity per cell in the Stem CellProliferation Assay cell population by at least 10%.

218. The composition of any of embodiments 212-217 wherein the at leastone Stem Cell Proliferator comprises at least one of a Stemness Driverand a Differentiation Inhibitor.

219. The composition of any of embodiments 212-218, wherein the at leastone Stem Cell Proliferator comprises both a Stemness Driver and aDifferentiation Inhibitor.

220. The composition of any of embodiments 212-219, wherein the StemCell Proliferator comprises a Stemness Driver in a concentration that is100-fold greater than an Effective Stemness Driver Concentration and aDifferentiation Inhibitor in a concentration that is at least 100-foldgreater than an Effective Differentiation Inhibition Concentration ofthe Differentiation Inhibitor.

221. The composition of any of embodiments 212-220, wherein thecomposition is a controlled release formulation.

222. The composition of embodiment 221 wherein the controlled releaseformulation when administered to a subject trans-tympanically imparts animmediate release, a delayed release, a sustained release, an extendedrelease, a variable release, a pulsatile release, or a bi-modal releaseof the stem cell proliferator.

223. The composition of embodiment 221 or 222 wherein the controlledrelease formulation when administered to a subject provides: (a) in anInitialPhase, at least an Effective Stemness Driver Concentration and atleast an Effective Differentiation Inhibition Concentration of theDifferentiation Inhibitor; and (b) in a Subsequent Phase, at least anEffective Stemness Driver Concentration and less than an EffectiveDifferentiation Inhibition Concentration of the DifferentiationInhibitor.

224. The method as in any of embodiments 191-211 or the composition asin any of embodiments 212-223 wherein the Stemness Driver is: aGSK3-beta inhibitor, a GSK3-beta inhibitor derivative, a Wnt agonist, aWnt agonist derivative, or a pharmaceutically acceptable salt of any ofthe foregoing.

225. The method as in any of embodiments 191-211 or the composition asin any of embodiments 212-223 wherein the Differentiation Inhibitor is:a notch agonist, a notch agonist derivative; an HDAC inhibitor; an HDACinhibitor derivative, or a pharmaceutically acceptable salt of any ofthe foregoing.

226. The method as in any of embodiments 191-211 or the composition asin any of embodiments 212-223, wherein the Stemness Driver is selectedfrom the group consisting of CHIR99021, LY2090314, lithium, A1070722,BML-284 and SKL2001

227. The method as in any of embodiments 191-211 or the composition asin any of embodiments 212-223, wherein the Differentiation Inhibitor isa Notch agonist or an HDAC inhibitor selected from the group consistingof valproic acid, SAHA and Tubastatin A.

228. A method of treating a subject who has, or is at risk ofdeveloping, hearing loss, the method comprising trans-tympanicallyadministering to a cochlear tissue of the subject a compositioncomprising at least one Stem Cell Proliferator.

229. The method of embodiment 228, wherein the at least one Stem CellProliferator comprises at least one of a Stemness Driver and aDifferentiation Inhibitor.

230. The method of embodiment 228 or 229, wherein the at least one StemCell Proliferator comprises both a Stemness Driver and a DifferentiationInhibitor. CLAIMS

231. A method for expanding a population of cochlear cells in a cochleartissue comprising a parent population of cells, the method comprisingcontacting the cochlear tissue with a stem cell proliferator to form anexpanded population of cells in the cochlear tissue, wherein the stemcell proliferator is capable of (i) forming a proliferation assay finalcell population from a proliferation assay initial cell population overa proliferation assay time period in a stem cell proliferation assay and(ii) forming a differentiation assay final cell population from adifferentiation assay initial cell population over a differentiationassay time period in a stem cell differentiation assay wherein:

(a) the proliferation assay initial cell population has (i) aproliferation assay initial number of total cells, (ii) a proliferationassay initial number of Lgr5⁺ cells, (iii) a proliferation assay initialnumber of hair cells, (iv) a proliferation assay initial Lgr5⁺ cellfraction that equals the ratio of the proliferation assay initial numberof Lgr5⁺ cells to the proliferation assay initial number of total cells,and (v) a proliferation assay initial hair cell fraction that equals theratio of the proliferation assay initial number of hair cells to theproliferation assay initial number of total cells;

(b) the proliferation assay final cell population has (i) aproliferation assay final number of total cells, (ii) a proliferationassay final number of Lgr5⁺ cells, (iii) a proliferation assay finalnumber of hair cells, (iv) a proliferation assay final Lgr5⁺ cellfraction that equals the ratio of the proliferation assay final numberof Lgr5⁺ cells to the proliferation assay final number of total cellsand (v) a proliferation assay final hair cell fraction that equals theratio of the proliferation assay final number of hair cells to theproliferation assay final number of total cells;

(c) the differentiation assay initial cell population has (i) adifferentiation assay initial number of total cells, (ii) adifferentiation assay initial number of Lgr5⁺ cells, (iii) adifferentiation assay initial number of hair cells, (iv) adifferentiation assay initial Lgr5⁺ cell fraction that equals the ratioof the differentiation assay initial number of Lgr5⁺ cells to thedifferentiation assay initial number of total cells, and (v) adifferentiation assay initial hair cell fraction that equals the ratioof the differentiation assay initial number of hair cells to thedifferentiation assay initial number of total cells;

(d) the differentiation assay final cell population has (i) adifferentiation assay final number of total cells, (ii) adifferentiation assay final number of Lgr5⁺ cells, (iii) adifferentiation assay final number of hair cells, (iv) a differentiationassay final Lgr5⁺ cell fraction that equals the ratio of thedifferentiation assay final number of Lgr5⁺ cells to the differentiationassay final number of total cells, and (v) a differentiation assay finalhair cell fraction that equals the ratio of the differentiation assayfinal number of hair cells to the differentiation assay final number oftotal cells;

(e) the proliferation assay final number of Lgr5⁺ cells exceeds theproliferation assay initial number of Lgr5⁺ cells by a factor of atleast 10; and

(f) the differentiation assay final number of hair cells is a non-zeronumber.

232. The method of embodiment 231, wherein the proliferation assay finalnumber of Lgr5⁺ cells is greater than the proliferation assay initialnumber of Lgr5⁺ cells by a factor of at least 50.

233. The method of embodiment 231, wherein the proliferation assay finalnumber of Lgr5⁺ cells is greater than the proliferation assay initialnumber of Lgr5⁺ cells by a factor of at least 100.

234. The method as in any of embodiments 231-233, wherein the expandedpopulation of cells in the cochlear tissue comprises a greater number ofhair cells than does the parent population.

235. The method as in any of embodiments 231-234, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5⁺ cell fraction by at least a factor of2.

236. The method as in any of embodiments 231-235, wherein thedifferentiation assay final hair cell fraction is greater than thedifferentiation assay initial hair cell fraction by at least a factor of2.

237. The method as in any of embodiments 231-236, wherein theproliferation assay final hair cell fraction is at least 25% less thanthe proliferation assay initial hair cell fraction

238. The method as in any of embodiments 231-237, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 10% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

239. The method as in any of embodiments 231-238, wherein the cochleartissue maintains Native Morphology.

240. The method as in any of embodiments 231-238, wherein the at leastone stem cell proliferator is dispersed in a biocompatible matrix.

241. The method as in any of embodiments 231-240, wherein theproliferation assay final number of Lgr5⁺ cells is greater than theproliferation assay initial number of Lgr5⁺ cells by a factor of atleast 100.

242. The method as in any of embodiments 231-240, wherein theproliferation assay final number of Lgr5⁺ cells is greater than theproliferation assay initial number of Lgr5⁺ cells by a factor of atleast 500.

243. The method as in any of embodiments 231-242, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5⁺ cell fraction by at least a factor of2.

244. The method as in any of embodiments 231-242, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5⁺ cell fraction by at least a factor of4.

245. The method as in any of embodiments 231-242, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5⁺ cell fraction by at least a factor of8.

246. The method as in any of embodiments 231-242, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5⁺ cell fraction by at least a factor of16.

247. The method as in any of embodiments 231-242, wherein theproliferation assay final Lgr5⁺ cell fraction is greater than theproliferation assay initial Lgr5+ cell fraction by at least a factor of32.

248. The method as in any of embodiments 231-247, wherein theproliferation assay final hair cell fraction is at least 25% less thanthe proliferation assay initial hair cell fraction.

249. The method as in any of embodiments 231-247, wherein theproliferation assay final hair cell fraction is at least 50% less thanthe proliferation assay initial hair cell fraction.

250. The method as in any of embodiments 231-247, wherein theproliferation assay final hair cell fraction is at least 75% less thanthe proliferation assay initial hair cell fraction.

251. The method as in any of embodiments 231-250, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 10% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

252. The method as in any of embodiments 231-250, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 10% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

253. The method as in any of embodiments 231-250, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 20% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

254. The method as in any of embodiments 231-250, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 30% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

255. The method as in any of embodiments 231-250, wherein theproliferation assay final Lgr5⁺ cell fraction is at least 50% greaterthan proliferation assay initial Lgr5⁺ cell fraction.

Further Embodiments Include

A method for expanding a population of LGR5+ cells in a cochlear tissuehaving an LGR5+ cell population, comprising:

contacting the cochlear tissue with a composition comprising at leastone Stem Cell Proliferator.

The method of Claim 1, wherein the at least one Stem Cell Proliferatoris capable, in a Stem Cell Proliferation Assay, of expanding an initialtest population of LGR5+ cells to create an expanded test population,wherein the expanded test population has at least 10-fold more LGR5+cells than does the initial test population.

The method of Claim 2, wherein the expanded test population has at least2-fold more Differentiation-Capable LGR5+ cells than does the initialtest population.

The method of Claim 2, wherein the expanded test population has at least10-fold more Differentiation-Capable LGR5+ cells than does the initialtest population.

The method as in any of Claims 1-4, wherein one of more MorphologicalCharacteristics of the cochlear tissue are maintained.

The method as in any of Claims 1-5, wherein the at least one Stem CellProliferator is dispersed in a Biocompatible Matrix.

The method of Claim 6, wherein the Biocompatible Matrix is aBiocompatible Gel or Foam.

The method as in any of Claims 1-7, wherein the composition is acontrolled release formulation.

The method as in any of Claims 1-8, wherein the cochlear tissue is an invivo cochlear tissue.

The method as in any of Claims 1-8, wherein the cochlear tissue is an exvivo cochlear tissue.

The method as in any of Claims 1-10, wherein the at least one Stem CellProliferator comprises at least one of a Stemness Driver and aDifferentiation Inhibitor.

The method of Claim 11, wherein the at least one Stem Cell Proliferatorcomprises both a Stemness Driver and a Differentiation Inhibitor.

The method of Claim 12, wherein the contacting provides:

in an Initial Phase, at least an Effective Proliferation Concentrationof the Stemness Driver and at least an Effective DifferentiationInhibition Concentration of the Differentiation Inhibitor; and

in a Subsequent Phase, at least an Effective Proliferation Concentrationof the Stemness Driver and less than an Effective DifferentiationInhibition Concentration of the Differentiation Inhibitor.

The method as in any of Claims 1-9 or 11-13, wherein the cochlear tissueis in a subject, and wherein the contacting the cochlear tissue with thecomposition is achieved by administering the compositiontrans-tympanically to the subject.

The method of Claim 14, wherein the contacting the cochlear tissue withthe composition results in improved Auditory Functioning of the subject.

A composition comprising:

at least one Stem Cell Proliferator, wherein the at least one Stem CellProliferator is capable, in a Stem Cell Proliferation Assay, ofexpanding an initial test population of LGR5+ cells to create anexpanded test population, and wherein the expanded test population hasat least 10-fold more LGR5+ cells than does the initial test population.

The composition of Claim 16, wherein the expanded test population has atleast 2-fold more Differentiation-Capable LGR5+ cells than does theinitial test population. [Please confirm that this is an appropriatefactor.]

The composition of Claim 16, wherein the expanded test population has atleast 10-fold more Differentiation-Capable LGR5+ cells than does theinitial test population.

The composition of Claim 16 or 18, wherein the at least one Stem CellProliferator is dispersed in a Biocompatible Matrix.

The composition of Claim 19, wherein the Biocompatible Matrix is aBiocompatible Gel or Foam.

A composition comprising:

at least one Stem Cell Proliferator dispersed in a Biocompatible MatrixSuitable For Cochlear Administration.

The composition as in any of Claims 16-21, wherein the at least one StemCell Proliferator comprises at least one of a Stemness Driver and aDifferentiation Inhibitor.

The composition as in any of Claims 16-22, wherein the at least one StemCell Proliferator comprises both a Stemness Driver and a DifferentiationInhibitor.

The composition as in any of Claims 16-23, wherein the composition is acontrolled release formulation.

The composition of Claim 24, wherein the controlled release formulationwhen administered to a subject trans-tympanically imparts an immediaterelease, a delayed release, a sustained release, an extended release, avariable release, a pulsatile release, or a bi-modal release of the stemcell proliferator.

The composition of Claim 24 or 25, wherein the controlled releaseformulation when administered to a subject provides: (a) in an InitialPhase, at least an Effective Proliferation Concentration of the StemnessDriver and at least an Effective Differentiation InhibitionConcentration of the Differentiation Inhibitor; and (b) in a SubsequentPhase, at least an Effective Proliferation Concentration of the StemnessDriver and less than an Effective Differentiation InhibitionConcentration of the Differentiation Inhibitor.

The method as in any of Claims 11-15 or the composition as in any ofClaims 16-26, wherein the Stemness Driver is: a GSK3-beta inhibitor, aGSK3-beta inhibitor derivative, a Wnt agonist, a Wnt agonist derivative,or a pharmaceutically acceptable salt of any of the foregoing.

The method as in any of Claims 11-15 or 27, or the composition as in anyof Claims 16-26 or 27, wherein the Differentiation Inhibitor is: a notchagonist, a notch agonist derivative; an HDAC inhibitor; an HDACinhibitor derivative, or a pharmaceutically acceptable salt of any ofthe foregoing.

The method of Claim 27 or 28 or the composition of Claim 27 or 28,wherein the Stemness Driver is selected from the group consisting of:CHIR99021, LY2090314, lithium, A1070722, BML-284 and SKL2001.

The method as in any of Claims 27-29 or the composition as in any ofClaims 27-29, wherein the Differentiation Inhibitor is a Notch agonistor an HDAC inhibitor selected from the group consisting of valproicacid, SAHA and Tubastatin A.

A method of treating a subject who has, or is at risk of developing,hearing loss, the method comprising:

trans-tympanically administering to a cochlear tissue of the subject acomposition comprising at least one Stem Cell Proliferator.

The method of Claim 31, wherein the at least one Stem Cell Proliferatorcomprises at least one of a Stemness Driver and a DifferentiationInhibitor.

The method of Claim 31 or 32, wherein the at least one Stem CellProliferator comprises both a Stemness Driver and a DifferentiationInhibitor.

A proliferating Lgr5 post-natal mammalian inner ear cell population thatexpresses at least one of the following compared to thenon-proliferating native state: reduced histone deacetylase, higherNotch, optionally wherein: where the cell in a supporting cell; wherethe supporting cell is Lgr5+; where notch agonist is an HDAC inhibitor;where a Wnt agonist is also included; where Wnt agonist is a GSK3-betainhibitor; and/or where the GSK3-beta inhibitor is CHIR99021

A method of administering to the ear of the subject a compositioncomprising VPA.

A method of administering to the ear of the subject a compositioncomprising CHIR99021,

A method of administering to the ear of the subject a compositioncomprising VPA and CHIR99021

A method to increase the ratio of hair cells to Lgr5⁺ support cells byproviding an agonist of the Wnt pathway and an agonist of notch pathwayor an antagonist of histone deacetylase, optionally where: WNTactivation is achieved by providing one or more antagonists of GSK3b;and/or the antagonist of histone deacetylase or Notch activation isachieved by providing VPA

A Method to expand Lgr5 cells by at least 3× and/or upregulate Lgr5expression by at least 3× by providing one or more Wnt agonists and oneor more antagonists of histone deacetylase or notch agonists.

A Method for expanding inner ear precursor cells by contacting the cellswith one or more agonists of Notch or antagonists of histone deacetylaseand an agonist of Wnt such that the cell proliferates, optionally where:in vitro the cell is exposed to additional growth factors; and/oradditional factors include the one or more agonist of Notch orantagonists of histone deacetylase and an agonist of Wnt areadministered in vivo to achieve a transient proliferation response.

An in vitro differentiated population of hair cells obtained fromculture-expanded Lgr5 cells with one or more Notch agonists and/orantagonists of histone deacetylase and/or one or more wnt agonists.

A Method for differentiation of Lgr5 precursor cells comprisingcontacting the cell with an amount of Notch inhibitor and CHIR99021,optionally where differentiation is into hair cells

A Method for differentiation of Lgr5 precursor cells into hair cellscomprising contacting the Lgr5 expressing cells with CHIR99021 and aNotch antagonist.

Any composition herein with the addition of antagonist of ROS to enhanceLgr5 expression and/or cell number expansion.

Any composition herein where Notch agonist is an HDAC inhibitor.

Any composition herein where HDAC inhibitor is VPA

Any composition herein where Wnt agonist is CHIR99021.

Delivery of CHIR to ear to expand and/or increase expression of Lgr5+supporting cells

Delivery of CHIR to the ear to increase the number of hair cells

Delivery of HDAC inhibitor (e.g VPA) to ear to expand and/or increaseexpression of Lgr5+ supporting cells

Delivery of both an HDAC inhibitor (e.g. VPA) and CHIR99021 to ear toincrease expression of Lgr5+ supporting cells

Any composition herein that is delivered to the middle and/or inner ear.

Any composition herein where Notch agonist or antagonists of histonedeacetylase are delivered in a pulsatile manner and Wnt agonist isdelivered in a sustained manner.

A method of treating or preventing hearing loss through administrationof CHIR99021 in a pharmaceutically relevant vehicle, optionally wherethe vehicle used herein is saline.

Any composition herein that is administered to the middle ear and/orinner ear

A method where tympanic membrane and/or surrounding tissue isanesthetized, a needle is placed into the middle ear, and agentsdescribed herein are injected.

A method to enhance transport of agents described herein in ear viapermeation enhancers, ultrasound, electroporation, and other methodsdescribed to those described in the art.

Any methods described herein be used in combination with agents that canincrease survival of supporting cells and or hairs cells.

Any suitable agent described herein delivered to a patient to enabledelivery of higher concentrations of drugs that are associated withdose-limiting ototoxicity.

A method where cells produced in vitro from agents or methods describedherein are used for research purposes and/or high throughput screening.

Methods or compositions where screening is used to identify agents toboost proliferation of Lgr5+ supporting cells, and or to test toxicityof drugs against supporting cells and/or their progeny, and or to testability of agents to improve survival of hair cells.

Any compositions herein that also includes at least one protective drug(that can enhance survival or prevent death of cells in the inner earincluding but not limited to hair cells)

A kit comprising: a container comprising, in a pharmaceuticallyacceptable carrier an inner-ear-supporting-cell-proliferation-inducingamount of Notch activators and/or HDAC inhibitors in combination withGSK3β inhibitors; and instructions for using the contents of containerto treat an inner ear disorder.

A method of treating or preventing hearing loss through administrationof a GSK3b inhibitor in a pharmaceutically relevant vehicle.

A method of producing Atoh-1+ in inner ear cells by treatment withCHIR99021, optionally where inner ear cells are Lgr5+ and differentiatein presence of CHIR99021 to produce Atoh-1+ cells and/or where Atoh-1+cells are hair cells.

A method of identifying a candidate agent that promotes proliferation ofepithelial stem cells that can differentiate to expresses atoh-1.

A method of identifying a candidate agent that promotes differentiationof epithelial stem cells that can express expresses atoh-1.

A method of identifying a candidate agent that promotes survival ofepithelial stem cells or atoh-1 expressing cells.

Additional Remarks Regarding Methodologies

Common methods for experimentation.

Those skilled in the art will recognize that there are manymethodologies for applying, testing, and treating with the agentsdescribed herein. Some non-limiting examples are described below.

Animals

For the experiments using inner ear spheres, C57BL/6 (The JacksonLaboratory) or Atoh1-nGFP reporter mice (Lumpkin et al., 2003) (a giftfrom Jane Johnson, University of Texas) of both sexes were used. Tocreate organ of Corti explants with ablated hair cells, we crossedMos-iCsp3 mice (line 17) (Fujioka et al., 2011) with Pou4f3-Cre mice(Sage et al., 2005) (a gift from Douglas Vetter, Tufts University). Forall in vivo experiments, we used a Cre reporter line, mT/mG (The JacksonLaboratory), crossed to a Sox2-CreER mouse (Arnold et al., 2011) (a giftfrom Konrad Hochedlinger, Mass. General Hospital) at 4 weeks of age.After genotyping, double-transgenic animals were used for lineagetracing. We used young adult wild-type littermates of the mT/mG;Sox2-CreER mice to prevent strain effects in the response to noise,which are known to vary depending on background (Harding et al., 2005;Wang et al., 2002). Mice were genotyped by PCR. All protocols wereapproved by the Institutional Animal Care and Use Committee ofMassachusetts Eye and Ear Infirmary or the by the ethics committee ofKeio University Union on Laboratory Animal Medicine, in compliance withthe Public Health Service policy on humane care and use of laboratoryanimals.

Isolation of Inner Ear Spheres

The utricles and cochleae of 1- to 3-day-old postnatal mice of bothsexes were dissected and, after careful removal of the nerve trunk andmesenchymal tissues, were trypsinized and dissociated. Dissociated cellswere centrifuged, and the pellet was resuspended and filtered through a70 m cell strainer (BD Biosciences Discovery Labware) in DMEM/F12 mediumwith N2/B27 supplement, EGF (20 ng/ml), IGF1 (50 ng/ml), bFGF (10ng/ml), and heparan sulfate (50 ng/ml) (Sigma). The single cells werecultured in nonadherent Petri dishes (Greiner Bio-One) to initiateclonal growth of spheres (Martinez-Monedero et al., 2008). Spheres thatformed after 2-3 days in culture were passaged every 4-6 days. Thespheres were centrifuged, and the pellet was mechanically dissociatedwith a pipette tip and resuspended in medium. Passage 3-4 spheres wereused for experiments described here. These cells are negative for haircell markers (Oshima et al., 2007) before the initiation ofdifferentiation. For differentiation, floating spheres were transferredto fibronectin-coated 4-well plates (Greiner Bio-One) as describedbefore (Martinez-Monedero et al., 2008; Oshima et al., 2007). Attachedspheres were differentiated for 5-7 days in DMEM/F12 medium with N2/B27supplement but without growth factors. γ-secretase inhibitors, DAPT,L-685458, MDL28170 (Sigma), and LY411575 (Santa Cruz) were added atseveral concentrations on the day after cell attachment.

Neonatal Cochlear Explants

Cochleae of 3-day-old postnatal C57BL/6 or Mos-iCsp3; Pou4f3-Credouble-transgenic mice of both sexes were dissected in Hanks solution(Invitrogen). To obtain a flat cochlear surface preparation, we removedthespiral ganglion, Reissner's membrane, and the most basal cochlearsegment. Explants were plated onto 4-well plates (Greiner Bio-One)coated with poly-L-ornithine (0.01%, Sigma) and laminin (50 μg/ml,Becton Dickinson). Cochlear explants were cultured in DMEM (Invitrogen)with 10% fetal bovine serum. All cultures were maintained in a 5%CO2/20% O2-humidified incubator (Forma Scientific).

Acoustic Overexposure

Four-week-old mice were exposed free field, awake and unrestrained, in asmall reverberant chamber (Wang et al., 2002). Acoustic trauma wasproduced by a 2 hr exposure to an 8-16 kHz octave band noise presentedat 116 dB SPL. The exposure stimulus was generated by a custom whitenoise source, filtered (Brickwall Filter with a 60 dB/octave slope),amplified (Crown power amplifier), and delivered (JBL compressiondriver) through an exponential horn fitted securely to a hole in the topof a reverberant box. Sound exposure levels were measured at fourpositions within each cage using a 0.25 inch Bruel and Kjor condensermicrophone: sound pressure was found to vary by <0.5 dB across thesemeasurement positions.

Systemic or Round Window Administration of Target Agent (e.g. LY411575)

Four-week-old mice weighing 12-16 g were used. Before surgery, theanimals were anesthetized with ketamine (20 mg/kg, intraperitoneally[i.p.]) and xylazine (100 mg/kg, i.p.), and an incision wasmadeposterior to the pinna near the external meatus after localadministration of lidocaine (1%). The otic bulla was opened to approachthe round window niche. The end of a piece of PE 10 tubing (BectonDickinson) was drawn to a fine tip in a flame and gently inserted intothe round window niche. LY411575 was dissolved in DMSO and diluted10-fold in polyethylene glycol 400 (Sigma) to a final concentration of 4mM. This solution (total volume 1 μl) was injected into the round windowniche of the left ear. Polyethylene glycol 400 with 10% DMSO wasinjected into the right ear as a control. The solution was administeredfor 2 min. This approach is widely used clinically and has the advantageof sparing the inner ear but still taking advantage of the local routeprovided by the round window membrane for delivery of drug into theinner ear (Mikulec et al., 2008). Gelatin was placed on the niche tomaintain the solution, and the wound was closed.

For the systemic administration, LY411575 (50 mg/kg) dissolved in 0.5%(w/v) methylcellulose (WAKO) was injected orally once daily for 5consecutive days. Hearing was measured by ABR at 1 day before, 2 days, 1week, 2 weeks, and 1, 2, and 3 months after noise exposure.

qRT-PCR

The organs of Corti were dissected in HBSS (Invitrogen) and stored inRNAlater (Ambion) at −80° C. until further use. Total RNA was extractedusing the RNeasy Mini Kit (QIAGEN) according to the manufacturer'sinstructions. For reverse transcription, SuperScript II (Invitrogen) wasused with random hexamers. The reverse transcription conditions were 25°C. for 10 min followed by 37° C. for 60 min. The reaction was terminatedat 95° C. for 5 min. cDNAs were mixed with Taqman Gene ExpressionMastermix (Applied Biosystems) and Hes5, Atoh1, or 18S primers (AppliedBiosystems) according to the manufacturer's instructions. Samples wereanalyzed in 96 wells in triplicate by qPCR (Applied Biosystems 7900HT),and PCR thermal cycling conditions were as follows: initial denaturationat 95° C. for 2 min, denaturation at 95° C. for 15 s, and annealing andextension at 60° C. for 1 min for 45 cycles. Conditions were keptconstant for each primer. Each PCR reaction was carried out intriplicate. Relative gene expression was analyzed by using the ΔΔCTmethod. Gene expression was calculated relative to 18S RNA, and theamount of cDNA applied was adjusted so that the Ct value for 18S RNA wasbetween 8 and 11.

Immunohistochemistry

For spheres, cells were fixed for 10 min with 4% paraformaldehyde inPBS. Immunostaining was initiated by blocking for 1 hr with 0.1% TritonX-100 in PBS supplemented with 1% BSA and 5% goat serum (PBT1). Fixedand permeabilized cells were incubated overnight in PBT1 with polyclonalantibody to myosin VIIa (Proteus Biosciences). Samples were washed threetimes for 20 min with PBS. Primary antibodies were detected withsecondary antibodies conjugated with Alexa 488 (Molecular Probes), withsecondary antibody alone used as a negative control. The samples werecounterstained with DAPI (Vector Laboratories) or Hoechst 33258(Invitrogen) for 10 min and viewed by epifluorescence microscopy(Axioskop 2 Mot Axiocam, Zeiss).

For explants, the organs of Corti were fixed for 15 min with 4%paraformaldehyde in PBS. Immunostaining was initiated by blocking thetissues for 1 hr with 0.1% Triton X-100 in PBS supplemented with 5%donkey serum (PBT1). Fixed and permeabilized pieces were incubatedovernight in PBT1 with antibodies to myosin VIIa (Proteus Biosciences),Sox2 (Santa Cruz), GFP (Invitrogen), prestin (Santa Cruz), neurofilamentH (Chemicon), and CtBP2 (BD Biosciences). Samples were washed threetimes for 20 min with PBS. Primary antibodies were detected withsecondary antibodies conjugated with Alexa 488 and 647 (MolecularProbes). The samples were stained with rhodamine phalloidin (Invitrogen)for 15 min and viewed by confocalfluorescence microscopy (TCS SP5,Leica).

For collection of the mature cochlea, deeply anesthetized mice weretranscardially perfused with 0.01 M phosphate buffer (pH 7.4) containing8.6% sucrose, followed by fixative consisting of freshly depolymerized4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Afterdecapitation, the temporal bones were removed and immediately placed inthe same fixative at 4° C. Small openings were made at the round window,oval window, and apex of the cochlea. After immersion in the fixativeovernight at 4° C., temporal bones were decalcified in 0.1 M EDTA (pH7.4) containing 5% sucrose with stirring at 4° C. for 2 days. Afterdecalcification, the cochlea was microdissected into four pieces forwhole-mount preparation. Immunostaining was initiated by blocking thetissues for 1 hr with 0.1% Triton X-100 in PBS supplemented with 5%donkey serum (PBT1). Fixed and permeabilized pieces were incubatedovernight in PBT1 with antibodies to myosin VIIa (Proteus Biosciences),Sox2 (Santa Cruz), and GFP (Invitrogen). Samples were washed three timesfor 20 min with PBS. Primary antibodies were detected with secondaryantibodies conjugated with Alexa 488, 568, and 647 (Molecular Probes)and viewed by confocal fluorescence microscopy (TCS SP5, Leica).Cochlear lengths were obtained for each case, and a cochlear frequencymap computed to precisely localize inner hair cells from the 5.6, 8.0,11.3, 16.0, 22.6, 32, and 45.2 kHz regions. For cross-sectioning, fixedtemporal bones were sunk in 30% sucrose in PBS at 4° C., incubated inOCT at room temperature for 1 hr, and frozen in liquid nitrogen. Thestaining protocol was the same as described above except forcounterstaining with DAPI (Vector Laboratories). Specimens were viewedby epifluorescence microscopy (Axioskop 2 Mot Axiocam, Zeiss).

Cell Counts

Cell counting for spheres was performed with MetaMorph software. Thecell number was determined from DAPI- or Hoechst-positive nuclei. Repeatcell counting gave a test variation of <1%. For explants, inner haircells, outer hair cells, and supporting cells in the outer hair cellregion were counted on cochlear whole mounts. Hair cells were identifiedwith myosin VIIa antibodies or endogenous GFP in Atoh1-nGFP mice.High-power images of the full-length cochlea or cochlear explantcultures were assembled and analyzed in PhotoShop CS4 (Adobe). ImageJsoftware (NIH) was used to measure the total length of cochlear wholemounts and the length of individual counted segments. The total numberof inner hair cells, outer hair cells, and supporting cells in the outerhair cell region was counted in each of four cochlear segments of1,200-1,400 m (apical, midapical, midbasal, and basal). Density (cellsper 100 m) was then calculated for each segment. For mature cochleae,high-power images of frequency-specific regions (5.6, 8.0, 11.3, and16.0 kHz) according to the computed frequency map were assembled andanalyzed. The number of inner hair cells, outer hair cells, andsupporting cells in the outer hair cell region in 100 m was counted ineach of the four frequency-specific regions of the cochlea. The numberof Sox2 lineage-positive cells identified by GFP was counted by the samemethod.

ABR Measurements

Auditory brain stem responses (Kujawa and Liberman, 1997; Maison et al.,2003) were measured in each animal at seven log-spaced frequencies(half-octave steps from 5.6 to 45.2 kHz) before and 1 day after noiseexposure, and 1 week, 1 month, and 3 months after surgery. Mice wereanesthetized with ketamine (100 mg/kg, i.p.) and xylazine (20 mg/kg,i.p.). Needle electrodes were inserted at vertex and pinna, with aground near the tail. ABRs were evoked with 5 ms tone pips (0.5 msrise-fall with a cos 2 onset envelope delivered at 35/s). The responsewas amplified, filtered, and averaged in a LabVIEW-driven dataacquisition system. Sound level was raised in 5 dB steps from >10 dBbelow threshold to <80 dB SPL. At each sound level, 1,024 responses wereaveraged (with stimulus polarity alternated), using an “artifactreject,” whereby response waveforms were discarded when peak-to-peakresponse amplitude exceeded 15 V. On visual inspection of stackedwaveforms, “ABR threshold” was defined as the lowest SPL level at whichany wave could be detected, usually corresponding to the level step justbelow that at which the peak-to-peak response amplitude rosesignificantly above the noise floor (approximately 0.25 μV). When noresponse was observed at the highest sound level available, thethreshold was designated as being 5 dB greater than that level so thatstatistical tests could be done. For amplitude versus level functions,the wave I peak was identified by visual inspection at each sound leveland the peak-to-peak amplitude was computed.

Quantification and Statistical Analysis

The two-tailed Mann-Whitney U test was used to compare differences amongtreatment groups. Changes before and after treatment of the same animalwere analyzed by two-tailed Wilcoxon t test. Repeated-measures ANOVA wasused to compare time-dependent differences among groups. Data arepresented in the text and in figures as mean SEM. p values less than0.05 were considered significant.

Genotyping Primers

We used the following genotyping primers: LacZ F:5′-ttcactggccgtcgttttacaacgtcgtga-3′ (SEQ ID NO: 1) and LacZ R:5′-atgtgagcgagtaacaacccgtcggattct-3′ (SEQ ID NO: 2) for the Mos-iCsp3mice; Cre F: 5′-tgggcggcatggtgcaagtt-3′ (SEQ ID NO: 3) and Cre R:5′-cggtgctaaccagcgttttc-3′(SEQ ID NO: 4) for the Pou4f3Cre and Sox2CreERmice; and oIMR7318 wild-type F: 5′-ctctgctgcctcctggcttct-3′ (SEQ ID NO:5), oIMR7319 wild-type R: 5′-cgaggcggatcacaagcaata-3′ (SEQ ID NO: 6),and oIMR7320 mutant R: 5′-tcaatgggcgggggtcgtt-3′ (SEQ ID NO: 7) for themT/mG mice.

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EXEMPLIFICATION Example 1

Cell culture: Heterozygous Lgr5-EGFP-IRES-CreERT2 mice were obtainedfrom Jackson Labs, and neonatal P2-P5 mice were used for cell isolation.Organ of Corti was isolated from Lgr5-GFP mice and further dissociatedinto single cells using trypsin. Cells were then cultured as previouslydescribed (Yin et al, 2014). Briefly, cells were entrapped in Matrigeland plated at the center of wells in a 24-well plate. Followingpolymerization of Matrigel, 500 μl of culture media (Advanced DMEM/F12with N2 and B27) was added, containing growth factors including EGF (50ng/ml) (epidermal growth factor), bFGF (20 ng/ml) (fibroblast growthfactor), and IGF1 (50 ng/ml) (insulin-like growth factor 1), and smallmolecules including CHIR99021 (5 μM), valproic acid (1 mM),2-phospho-L-ascorbic acid (280 μM), and 616452 (2 μM). Y-27632 (10 μM)was added for the first 2 days. In some experiments, bFGF was used at 50ng/ml.

For single cell isolation, the organs of Corti were then treated withCell Recovery Solution (Corning) for 1 h to separate cochlear epitheliumfrom the underlying mesenchyme. Epithelia were then collected andtreated with Trypsin (Life Technologies) for 15-20 minutes at 37° C.Single cells obtained by mechanical trituration were filtered by cellstrainer (40 μm) and washed with cell culture media. For FACS sorting ofLgr5-high and Lgr5-Low cells, FACS ARIA (BD) was used and Lgr5-GFP wasused as indicator of Lgr5 level. Sorted single cells were cultured inMatrigel in inner ear cell culture media with supplement growth factorsand small molecules. Y-27632 was added in the first 2 days.

FACS analysis: Cell culture media was removed and Trypsin was added.After incubation at 37° C. for 10-20 min, colonies were dissociated intosingle cells. Live-cell number were counted using a hemocytometer andtrypan blue staining. The cells were then stained with propidium iodide(PI) and analyzed with flow cytometry. The number of GFP-positive cellswas calculated by multiplying the total number of cells by thepercentage of GFP-positive cells.

Results

Lgr5 cells are present within a subset of supporting cells within thecochlear epithelium. Using an Lgr5-GFP mouse line, we tested theactivation or inhibition of multiple pathways to expand single Lgr5⁺supporting cells isolated from the cochlea in a Matrigel based 3Dculture system. Inner ear epithelial cells have been shown to be able tobe cultured as neuro-spheres in the presence of growth factors includingepidermal growth factor (EGF or E), basic fibroblast growth factor (bFGFor F), and insulin like growth factor 1 (IGF-1 or I) (Li et al., 2003).However, in this condition, no Lgr5-GFP cell growth was observed (FIG.1A). We initially tested a combination of small molecules including theglycogen synthase kinase 30 (GSK30) inhibitor, CHIR99021 (CHIR or C) andthe histone deacetylase (HDAC) inhibitor, valproic acid (VPA or V) withthe growth factor cocktail. We found CHIR and VPA greatly promoted theexpansion of Lgr5-GFP cells, and large colonies of Lgr5-GFP+ cells wereobserved in the culture (FIG. 1). Flow cytometry analysis revealed thecombination of CHIR and VPA greatly increased GFP+ cell percentage (FIG.2A). Similar phenotype was also observed when the inner ear Lgr5 cellswere cultured using culture condition that was previously used forintestinal LGR5+ cells (Yin et al., 2014), although to a less extend(FIG. 2B). Cell number quantification revealed that the addition of CHIRand VPA significantly increased cell proliferation and Lgr5-GFPexpression of the cells, resulting in a −500 fold increase in Lgr5-GFPcells (FIG. 2C).

Unlike the intestinal colonies, cells from the inner ear epithelium lostthe capacity for proliferation after passage. We reasoned that otherfactors were needed for prolonged culture of the cells and performedscreening to identify additional factors. Addition of2-phospho-L-ascorbic acid (pVc, Sigma), a stable form of vitamin C,increased Lgr5+ cell expansion by an additional 2-3 fold (FIG. 3 andFIG. 8B), which was further included in the culture media (EFI.C.V.P).We also found increased bFGF concentration (from 20 ng/ml to 50 ng/ml)increased cell proliferation (FIG. 4) thus bFGF was used at 50 ng/ml inthe future experiments. Adding TTNPB, an RAR agonist, slightly increasedcell proliferation but not GFP expression (FIGS. 5A-B), thus we didn'tinclude TTNPB in the culture condition for neonatal cells. Furtherscreen of small molecule modulators of major signaling pathwaysdemonstrated these signaling pathway modulators didn't increase Lgr5-GFPexpression (FIG. 6). Interestingly, we found that unlike the role of BMPinhibition in the small intestinal stem cells, where it's essential topromote the expression of Lgr5, the addition of BMP inhibitor LDN193189greatly decreased Lgr5-GFP expression (FIG. 6). In addition, althoughthe Tgf-β receptor (ALK5) inhibitor 616452 (Calbiochem, 6, also known asRepsox) didn't increase Lgr5-GFP expression, we observed that thecolonies in conditions with 616452 tend to grow larger and possessbrighter GFP expression than that in control conditions (FIG. 7).Further validation showed that the addition of 616452, also increasedcell expansion (by 2-3 fold) before and after passage, and enabled thepassage of colonies for up to 5 generations (FIG. 8B and FIG. 9F).Collectively, the addition of small molecules (CVP6) increased Lgr5+cell number by >2000 fold compared to growth factors alone (EFI) (FIGS.8A-C and FIGS. 9A-F).

To examine the relative importance of individual factors in our culturesystem (without passaging), we separately removed each factor from themedium and quantified cell proliferation and Lgr5 expression of innerear epithelial cells following 10 days of culture (FIG. 8B). Removal ofCHIR or bFGF had the greatest effect on proliferation, while removal ofCHIR had the greatest effect on Lgr5 expression. Removing EGF or 616452caused a significant reduction in proliferation, while removing VPA orpVc greatly reduced Lgr5 expression. The presence of IGF1 had a marginalbeneficial effect on cell proliferation and Lgr5 expression. Thetreatment with the combined agents (EFICVP6) yielded the highest numberof total cells, Lgr5+ cells and percentage of Lgr5+ cells following 10days of culture. These results suggest that bFGF and CHIR were mostcritical while the other factors promoted maximal proliferation and Lgr5expression. Similar results were obtained by direct visualization of GFPexpression and cell growth (FIG. 8C).

We further examined the potential function of individual factors. Theeffects of CHIR in promoting cell proliferation and Lgr5 expressioncould be partially replicated with Wnt3a in combination with R-spondin1(FIG. 9A), suggesting a role of CHIR in activating the Wnt pathway.Using an Atoh1-nGFP mouse line, we found that VPA suppressed spontaneousdifferentiation of hair cells (FIG. 9D), which is consistent with therole of VPA in maintaining Notch activation in intestinal stem cells(Yin et al., 2014).

To demonstrate that single sorted Lgr5-GFP cells can grow into GFP+colonies in our expansion condition (EFICVP6), we sorted single Lgr5-GFPcells from inner ear epithelial into GFP-high and GFP-low fractions.Following 14 days of expansion in EFICVP6 condition, cultures initiatedfrom single GFP-high cells contained high purity of colonies whichhighly express Lgr5-GFP. Whereas cultures initiated from GFP-low cellscontained both GFP-high and GFP-low and GFP negative colonies (FIG. 10).This experiment demonstrated single sorted Lgr5-GFP cells can beexpanded in EFICVP6 condition.

Example 2: Differentiation of Expanded Lgr5-Positive Cells to Hair Cells

Materials and Methods

Differentiation protocol: For differentiation of expanded Lgr5-positivecells, following 10 days of culture in the cell expansion condition(EFICVP6), the cell colonies were transferred to fresh Matrigel andfurther cultured in differentiation media. Differentiation mediacontains Notch pathway inhibitor (e.g. DAPT, D, 5 μM, or LY411575, LY, 5μM), with or without Gsk3β inhibitor (e.g. CHIR99021). Media werechanged every other day. Following another 6-10 days of incubation withdifferentiation media, the colonies were harvested for qPCR analysis orfixed with 4% PFA and immunostained with hair cell markers Myo7a andPrestin.

RNA extraction and quantitative real-time PCR (qPCR): RNA was isolatedfrom cultured cells (RNeasy Mini Kit; Qiagen) according to themanufacturer's protocol. Quantitative real-time PCR was performed withQuantiTect Probe PCR kit (Qiagen) using commercially available primersand TaqMan probes (Myo7a and Hprt Life Technologies).

Immunocytochemistry staining: Colonies were fixed at room temperature in4% paraformaldehyde/PBS for 15-20 min and then washed twice with PBScontaining 0.1% BSA. Cells were then permeabilized with 0.25% TritonX-100 in PBS in 4° C. for 30 minutes. Following 2 washes with PBScontaining 0.1% BSA, the cells were incubated with blocking solution(Power Block, Biogenex) for 1 h. Diluted primary antibodies (in PowerBlock solution) was applied for 4 h at room temperature or overnight at4° C. Primary antibodies used were Myosin VIIA (1:500, Rabbit polyclonalfrom Proteus Biosciences) and Prestin (1:400, Goat polyclonal from SantaCruz). After 3 washes for 5 minutes each, secondary antibodies(Alexafluor 594 and 647-conjugated; Invitrogen) were added at 1:500dilution and incubated at room temperature for 30 minutes. Nuclei werevisualized with 4,6-diamidino-2-phenylindole (DAPI, VectorLaboratories). Staining was visualized with inverted fluorescencemicroscope (EVOS; Advanced Microscopy Group).

Results

As an important function proof that the expanded Lgr5-positive cells arestem cells, we tested the differentiation ability of the expanded cellsin vitro. Notch inhibition was shown to promote the differentiation ofLgr5 supporting cells to hair cells in vivo (Jeon et al., 2011). Inaddition, Wnt pathway activation achieved by β-catenin expression hasalso been shown to promote Atoh1 expression and hair celldifferentiation (Shi et al., 2013). Thus, we tested these conditions ininducing hair cell differentiation of expanded Lgr5 cells.

We first tested multiple conditions with different combination of growthfactors (EGF, bFGF, IGF1) or small molecules (CHIR, VPA, 616452, pVc)and Notch inhibitor (e.g. LY411575) for the expression of hair cellmarker Myo7a. The condition without growth factors or small moleculesbut with CHIR and Notch inhibitor gave the highest increase of Myo7aexpression (FIG. 11), suggesting growth factors (EGF, bFGF, IGF1) orsmall molecules (VPA, 616452 and pVc) inhibit the differentiation ofhair cells and should be removed in the differentiation media.

We treated Lgr5-GFP cells, expanded by the above procedures with DAPT, aγ-secretase inhibitor and CHIR, the GSK3Beta inhibitor. Following 10days of differentiation, the generation of hair cells were visualized bystaining with hair cell markers including Myosin VIIA and Prestin. Thecombination of DAPT with CHIR induced hair cell generation indicated byMyosin VIIA and Prestin positive colonies (outer hair cells) (FIG. 12Aupper panels) and Myosin VIIA positive but Prestin negative colonies(inner hair cells) (FIG. 12A, lower panels). Without the activation ofWnt signaling by GSK3Beta inhibitor, or with the inhibition of Wntsignaling by small molecule Wnt pathway inhibitor (IWP-2, 2 μM), haircell generation is rarely observed (FIG. 12B).

Example 3: Expansion and Hair Cell Differentiation of Lgr5-ExpressingCells from Adult Inner Ear Tissue

Materials and Methods

For adult tissue, the stria vascularis was removed but the epitheliumwas not removed from the underlying mesenchyme due to the limited amountof intact cochlea that could be extracted. For adult cells, additionalsmall molecule TTNPB (2 μM, Tocris) was added and the complete mediacontain EGF, bFGF, IGF-1, CHIR99021, VPA, pVc, 616452 and TTNPB.

Results

We found that although the condition EFICVP6 support the survival andgrowth of Lgr5 inner ear cells from adult mice (FIGS. 13A-C), theproliferation was very slow. We thus performed addition screening foraddition factors that can promote the proliferation of Lgr5 cells fromadult mice. We found that a small molecule RA signaling pathway agonist,TTNPB, significantly promoted the proliferation of cultured cells (FIG.14). Thus it was further incorporated in the expansion media for Lgr5inner ear cells.

We tracked cell colonies formation of cells isolated from a 6-week oldadult mouse. After 5 days of culture in EFICVP6+TTNPB condition, singlecells form large GFP+ colonies confirming the proliferation of adultLgr5 cells in this condition (FIG. 15). From a single cochlea, a largenumber of GFP+ colonies can be expanded (FIG. 14).

Discussion of Examples 1-3

The experiments illustrated in FIGS. 1A-B, 2A-C, 3, 4, 5A-B, 6, 7, 8A-C,9A-F, 10, 11 12A-B, 13A-C, and 14 show the following:

FIGS. 1A-B and FIGS. 2A-C

Cocktail containing growth factors and small molecules including CHIRand VPA promote the proliferation and GFP expression of Lgr5 inner earprogenitor cells in vitro, and permit the expansion of these cells.

FIG. 3.

The addition of pVc (2-phospho-L-ascorbic acid) increases cellproliferation of Lgr5 inner ear progenitor cells

FIG. 4.

Increasing bFGF concentration promotes the proliferation of Lgr5 innerear progenitor cells. FIGS. 5A-B, 6, 7

Additional small molecule (616452) promotes the proliferation of Lgr5inner ear progenitor cells.

FIGS. 8A-C.

1. Cocktail containing growth factors and small molecules maintainsLgr5+ inner ear progenitors in vitro.

2. Cocktail containing all the factors (EGF, bFGF, IGF1, CHIR99021, VPA,pVc, 616452 (EFICVP6)) shows the best result in supporting cellproliferation and GFP expression, with 4.7×10⁵ total number of cells,58% GFP+ cells and 2.7×10⁵ GFP+ cells.

3. Most of the factors in the cocktail are important:

CHIR is important to promote cell proliferation and GFP expression:

Removing CHIR results in ˜100 fold decrease of total cell numbers(4.7×105 v.s. 5.0×103) and ˜50 fold decrease of GFP+ cells. (58% v.s.1.3%). With ˜4000 fold decrease of GFP+ cells (2.7×105 v.s. ˜65). bFGFis important to promote cell proliferation and important for GFPexpression: Removing bFGF results in 10 fold decrease of cell numbers.(4.2×104 total number of cells) and ˜2 fold decrease of GFP+ percentage(58% v.s. 32%) as well as 20 fold decrease of GFP+ cell number (2.7×10⁵v.s. 1.4×10⁴ GFP+ cells).

4. EGF, 616452 are important to promote cell proliferation. Removing EGFresults in ˜2 fold decrease of total cell number (2.2×105) and GFP+ cellnumber (1.1×105). Removing 616452 results in ˜3 fold decrease of totalcell number (1.7×10⁵) and GFP+ cell number (9.8×10⁴).

5. VPA and pVc are important to promote GFP maintenance. Removing VPAresults in 2 fold decrease of GFP percentage 28% and GFP+ cell number(1.1×105). Removing pVc results in 2 fold decrease of GFP percentage 25%and GFP+ cell number (1.1×10⁵).

6. IGF-1 has marginal effect to promote cell proliferation (4.1×10⁵total cells) or GFP maintenance (58% v.s. 53% GFP percentage).

FIGS. 9A-F

1. CHIR functions through Wnt pathway.

2. Combine with R-spondin1, Wnt3a may replace CHIR but not as effective.

3. VPA functions through suppressing differentiation (likely byactivating/maintain Notch activation).

4. HDAC6 inhibitor does not work when HDAC6 inhibitor is used in placeof VPA (Data not shown, trying Tubastatin A, ACY1215 and CAY10603).

5. pVc promotes GFP expression.

6. Laminin 511 promotes GFP expression.

7. Tgfp type I receptor (Tgf R1, ALK5) inhibitor 616452 enables extendedculture of the cells.

8. CHIR can promote differentiation to Atoh-1+ cells.

FIG. 10.

Single sorted Lgr5-GFP cells can be expanded in cocktails containinggrowth factors and small molecules.

FIG. 11

1. The presence of Wnt pathway/CHIR increases the differentiationefficiency of a Notch inhibitor.

2. Cultured Lgr5 progenitor cells can generate both inner hair cells andouter hair cells.

In some in vivo embodiments the growth factors are not required.

FIGS. 13A-C

1. The expansion protocol for LGR5+ cells works on adult cochlea frommice and the cells can be passaged without loosing Lgr5 expression.

FIGS. 14-15

Small molecule TTNPB promote the proliferation of adult Lgr5 inner earprogenitor cells.

Example 4: Poloxamer-407 Hydrogel for Delivery of Small Molecules toInner Ear for Regeneration of Hair Cells

1. Methods

1.1 Preparation of Formulation

Poloxamer-407 hydrogels were prepared using the “cold-method”. Briefly,a weighed amount of poloxamer-407 was added to 40 ml cold ultra purewater or cold PBS (pH 7.4), and stirred overnight at 4° C. on a magneticstir plate to effect complete solubilization. Multiple concentrations ofpoloxamer-407 solution ranging from 18% (w/w) to 25% (w/w) wereprepared. Hydrophilic drugs, including valproic acid (VPA) andphosphorylated ascorbic acid (PAC) were added to the 5 ml poloxamer-407solution and dissolved at 4° C. on a magnetic stir plate. Weight ratioof poloxamer-407 to the drug was varied to understand the effects ofdrugs on the gelation properties of poloxamer-407, and to determine theoptimal formulation that gels at 37° C. with maximum possible loading ofthe hydrophilic drugs. The gelation temperatures of the formulationswere determined by the “visual tube inversion method”. Briefly, glassvials containing poloxamer 407 solutions, with or without thehydrophilic drugs were placed in a water bath. The temperature wasslowly increased and the temperature at which the solution stoppedflowing on tilting the glass vial was noted as the gelation temperature.

To encapsulate hydrophobic drugs, including CHIR 99021 (CHIR), Repsoxand TTNPB, appropriate volumes from their stock solutions in DMSO wereadded into the poloxamer-407 solutions containing the hydrophilic drugs,and mixed by pipetting at 4° C. Maximum DMSO concentration to be addedwith the hydrophobic drugs was limited to 5-6% (v/v) with respect to thetotal volume of the gel. Higher concentration of DMSO reduced thegelation temperature of the gel. Gelation temperature of the formulationwas determined by the “visual tube inversion method”, as describedbefore.

1.2 In Vitro Drug Release

To understand the release kinetics of encapsulated drugs frompoloxamer-407 hydrogels, in vitro release studies were performed usingdialysis bag method at pH 7.4 and 37° C. temperature conditions.Briefly, sealed dialysis bag (3.5-5 kDa cutoff) containing 30 μL gelsuspended in 1 ml PBS was placed in 10 mL of the release medium (PBS).The release medium was stirred at 100 rpm to prevent the formation of astagnant layer at the membrane and outer solution interface. 1 mLaliquots were taken from the medium at predetermined intervals for theanalysis of drugs using HPLC, and replaced with an equal volume of freshmedium.

Results

2.1 Formulation and Gelation:

The thermal gelation behavior of different formulations was investigatedto determine the optimal formulation that would provide a rapid andreproducible liquid-gel transition between room and body temperatureswhile loaded with all the drugs. For poloxamer 407 solutions, withoutdrug, the gelation temperature decreased with increasing theconcentration of poloxamer 407. Addition of hydrophilic drugs, includingVPA and pVc, at concentrations greater than 88 mg/ml and 14 mg/ml,respectively, inhibited gelation of poloxamer-407 solution. Therefore,gels were prepared using 18% (w/w) poloxamer solutions withconcentrations of VPA and pVc to be equal to or less than 88 mg/ml and14 mg/ml, respectively. Hydrophobic drugs, including CHIR, Repsox andTTNPB, appropriate volumes from their stock solutions in DMSO were addedinto the poloxamer-407 solutions containing the hydrophilic drugs, andmixed by pipetting at 4° C. Concentrations of drugs in stock solutionswere maintained at 55.6-69.5 mg/ml, 23-28.75 mg/ml and 35 mg/ml forCHIR, Repsox and TTNPB, respectively to ensure total DMSO concentrationin final formulation to be less than 5-6%. Higher concentration of DMSOsignificantly lowered the gelation temperature of the formulations. Thefinal formulation was a viscous liquid at storage temperature (4° C.),and formed a semisolid gel above its liquid-gel transition temperature(37° C.).

2.2 In Vitro Drug Release:

In vitro release of encapsulated drugs from hydrogel formulation wasstudied using dialysis bag method. Both CHIR and VPA showed an initialburst release of 10% in 2 h and 17% in 0.5 h, respectively, followed bya sustained release over next 48 h. Also, the release kinetics of VPAwas found to be significantly faster as compared to that of CHIR.Quantitatively, 33% cumulative release was observed for CHIR in 48 h,while 98% cumulative release was observed for VPA in 48 h. The detailedrelease profiles are shown in FIGS. 16A-B and 17A-B.

Example 5: Hearing Recovery in Mice

A 17% (w/w) stock solution of poloxamer 407 gel (Sigma-Aldrich) wasprepared by slowly adding it to cold 1× phosphate-buffered saline at pH7.4. This solution is liquid when refrigerated or at room temperaturebut solidifies at body temperature. The gel was tinted blue with Evansblue dye (50 ppm) for visualization during administration.

A formulation was prepared as described in Example 4 with 527 mM VPA and2.975 mM CHIR in 17% Poloxamer 407 with 5% DMSO (“VPA/CHIR”). Forcomparison, a Notch inhibitor, LY411575, was prepared at 4 mM in 17%Poloxamer 407 with 5% DMSO (“LY411575”). Additionally, a vehicle-onlycontrol of 17% Poloxamer 407 with 5% DMSO was prepared (“Control”).

CBA/CaJ mice were deafened in a noise chamber by exposure to an 8-16 kHzoctave band noise band for 2 hours at 120 dB SPL. 24 hours after noiseexposure, their hearing was assessed by measuring auditory brainstemresponses (ABRs). The minimum sound pressure level (SPL) required forvisual detection of ABR Wave I was determined at 5, 10, 20, 28.3, and 40kHz. Following ABR measurements, a trans-tympanic injection of aPoloxamer gel drug mixture (described above as “VPA/CHIR”, “LY411575”,or “Control”) was delivered to fill the middle ear cavity. After 30days, ABR was assessed and the improvement in hearing threshold from 24hrs to 30 days after noise exposure was determined. The results areshown in FIG. 18.

A 10 dB improvement in threshold creates a doubling in loudness for agiven sound and is considered to be clinically meaningful. The“VPA/CHIR” formulation achieved a 10 dB recovery. Statisticallysignificant improvements are shown with a star (*means p<0.05).

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Suchembodiments are also within the scope of the following claims. Therecitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof. Theteachings of all patents, published applications and references citedherein are incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

What is claimed is:
 1. A method for expanding a population of cochlearcells in a cochlear tissue, the method comprising administeringintratympanically or transtympanically to a subject a compositioncomprising: i) a biocompatible matrix having dispersed therein: a) aGSK3β inhibitor or Wnt agonist, or a pharmaceutically acceptable saltthereof; and b) a Notch agonist or HDAC inhibitor, or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein the GSK313inhibitor or Wnt agonist, or pharmaceutically acceptable salt thereof,is selected from CHIR99021, LY2090314, lithium, A1070722, BML-284 andSKL2001, or a pharmaceutically acceptable salt thereof.
 3. The method ofclaim 1, wherein the GSK3β inhibitor or Wnt agonist, or pharmaceuticallyacceptable salt thereof, is:

or a pharmaceutically acceptable-salt thereof.
 4. The method of claim 1,wherein the GSK3β inhibitor or Wnt agonist is LY2090314, or apharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein the GSK3β inhibitor or Wnt agonist is lithium, or apharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein the GSK3β inhibitor or Wnt agonist is A1070722, or apharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the GSK3β inhibitor or Wnt agonist is BML-284, or apharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the GSK313 inhibitor or Wnt agonist is SKL2001, or apharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein the Notch agonist or HDAC inhibitor, or pharmaceuticallyacceptable salt thereof, is selected from valproic acid, SAHA andTubastatin A, or a pharmaceutically acceptable salt thereof.
 10. Themethod of claim 1, wherein the Notch agonist or HDAC inhibitor, orpharmaceutically acceptable salt thereof, is:

or a pharmaceutically acceptable salt thereof.
 11. The method of claim1, wherein: the GSK3β inhibitor or Wnt agonist, or pharmaceuticallyacceptable salt thereof, is selected from CHIR99021, LY2090314, lithium,A1070722, BML-284 and SKL2001, or a pharmaceutically acceptable saltthereof; and the Notch agonist or HDAC inhibitor, or pharmaceuticallyacceptable salt thereof, is selected from valproic acid, SAHA andTubastatin A, or a pharmaceutically acceptable salt thereof.
 12. Themethod of claim 1, wherein: the GSK3β inhibitor or Wnt agonist, orpharmaceutically acceptable salt thereof, is CHIR99021, or apharmaceutically acceptable salt thereof; and the Notch agonist or HDACinhibitor, or pharmaceutically acceptable salt thereof, is valproicacid, or a pharmaceutically acceptable salt thereof.
 13. The method ofclaim 1, wherein: the GSK3β inhibitor or Wnt agonist, orpharmaceutically acceptable salt thereof, is between about 0.01 wt % andabout 50 wt % of the composition; and the Notch agonist or HDACinhibitor, or pharmaceutically acceptable salt thereof, is between about0.01 wt % and about 50 wt % of the composition.
 14. The method of claim1, wherein: the GSK3β inhibitor or Wnt agonist, or pharmaceuticallyacceptable salt thereof, is between about 0.1 wt % and about 50 wt % ofthe composition; and the Notch agonist or HDAC inhibitor, orpharmaceutically acceptable salt thereof, is between about 0.1 wt % andabout 50 wt % of the composition.
 15. The method of claim 1, wherein:the GSK3β inhibitor or Wnt agonist, or pharmaceutically acceptable saltthereof, is between about 0.1 wt % and about 40 wt % of the composition;and the Notch agonist or HDAC inhibitor, or pharmaceutically acceptablesalt thereof, is between about 0.1 wt % to about 40 wt % of thecomposition.
 16. The method of claim 1, wherein: the GSK3β inhibitor orWnt agonist, or pharmaceutically acceptable salt thereof, is betweenabout 0.1 wt % and about 30 wt % of the composition; and the Notchagonist or HDAC inhibitor, or pharmaceutically acceptable salt thereof,is between about 0.1 wt % to about 30 wt % of the composition.
 17. Themethod of claim 1, wherein: the GSK3β inhibitor or Wnt agonist, orpharmaceutically acceptable salt thereof, is between about 0.1 wt % andabout 20 wt % of the composition; and the Notch agonist or HDACinhibitor, or pharmaceutically acceptable salt thereof, is between about0.1 wt % and about 20 wt % of the composition.
 18. The method of claim1, wherein: the GSK3β inhibitor or Wnt agonist, or pharmaceuticallyacceptable salt thereof, is between about 0.1 wt % and about 10 wt % ofthe composition; and the Notch agonist or HDAC inhibitor, orpharmaceutically acceptable salt thereof, is between about 0.1 wt % andabout 10 wt % of the composition.
 19. The method of claim 1, wherein:the GSK3β inhibitor or Wnt agonist, or pharmaceutically acceptable saltthereof, is between about 0.1 mg/mL to about 70 mg/mL of thecomposition; and the Notch agonist or HDAC inhibitor, orpharmaceutically acceptable salt thereof, is between about 0.01% toabout 50% the composition.
 20. The method of claim 1, wherein the GSK3βinhibitor or Wnt agonist, or pharmaceutically acceptable salt thereof,and the Notch agonist or HDAC inhibitor, or pharmaceutically acceptablesalt thereof, are in lyophilized form.
 21. The method of claim 1,wherein the GSK3β inhibitor or Wnt agonist, or pharmaceuticallyacceptable salt thereof, and the Notch agonist or HDAC inhibitor, orpharmaceutically acceptable salt thereof, are in hydrated form.
 22. Themethod of claim 1, wherein the biocompatible matrix comprises one ormore of hyaluronic acid, hyaluronates, lecithin gels, pluronics,poly(ethyleneglycol), poloxamers, chitosans, xyloglucans, collagens,fibrins, polyesters, poly(lactides), poly(glycolide),poly(lactic-co-glycolic acid (PLGA), sucrose acetate isobutyrate,glycerol monooleate, poly anhydrides, poly caprolactone sucrose, orglycerol monooleate or a combination thereof.
 23. The method of claim 1,wherein the biocompatible matrix comprises a poloxamer.
 24. The methodof claim 23, wherein the poloxamer is Poloxamer
 407. 25. The method ofclaim 1, wherein the biocompatible matrix comprises hyaluronic acid. 26.The method of claim 1, wherein the biocompatible matrix is a hydrogel.27. The method of claim 1, wherein the biocompatible matrix is abiocompatible gel or foam.
 28. The method of claim 1, wherein thecomposition is a controlled release formulation.
 29. The method of claim28, wherein the controlled release formulation when administered to asubject intratympanically or transtympanically imparts an immediaterelease, a delayed release, a sustained release, an extended release, avariable release, a pulsatile release, or a bi-modal release of one ormore of a) the GSK3β inhibitor or Wnt agonist, or a pharmaceuticallyacceptable salt thereof; and b) the Notch agonist or HDAC inhibitor, ora pharmaceutically acceptable salt thereof.
 30. The method of claim 1,wherein the method produces a population of Lgr5+ cells that are ins-phase.
 31. The method of claim 1, wherein administeringintratympanically or transtympanically the composition to a subjectresults in improved auditory functioning of the subject.
 32. The methodof claim 1, wherein administering is intratympanically.
 33. The methodof claim 1, wherein administering is transtympanically.
 34. A method forexpanding a population of cochlear cells in a cochlear tissue, themethod comprising administering intratympanically or transtympanicallyto a subject a composition comprising: i) a biocompatible matrix havingdispersed therein: a) a first compound selected from CHIR99021,LY2090314, lithium, A1070722, BML-284 and SKL2001, or a pharmaceuticallyacceptable salt thereof; and b) a second compound selected from valproicacid, SAHA and Tubastatin A, or a pharmaceutically acceptable saltthereof.
 35. The method of claim 34, wherein the first compound isCHIR99021, or a pharmaceutically acceptable salt thereof.
 36. The methodof claim 34, wherein the first compound is LY2090314, or apharmaceutically acceptable salt thereof.
 37. The method of claim 34,wherein the first compound is lithium, or a pharmaceutically acceptablesalt thereof.
 38. The method of claim 34, wherein the first compound isA1070722, or a pharmaceutically acceptable salt thereof.
 39. The methodof claim 34, wherein the first compound is BML-284, or apharmaceutically acceptable salt thereof.
 40. The method of claim 34,wherein the first compound is SKL2001, or a pharmaceutically acceptablesalt thereof.
 41. The method of claim 34, wherein the second compound isvalproic acid, or a pharmaceutically acceptable salt thereof.
 42. Themethod of claim 34, wherein: the first compound is CHIR99021, or apharmaceutically acceptable salt thereof; and the second compound isvalproic acid, or a pharmaceutically acceptable salt thereof.
 43. Themethod of claim 34, wherein: the first compound is between about 0.01 wt% and about 50 wt % of the composition; and the second compound isbetween about 0.01 wt % and about 50 wt % of the composition.
 44. Themethod of claim 34, wherein: the first compound is between about 0.1 wt% and about 50 wt % of the composition; and the second compound isbetween about 0.1 wt % and about 50 wt % of the composition.
 45. Themethod of claim 34, wherein: the first compound is between about 0.1 wt% and about 40 wt % of the composition; and the second compound isbetween about 0.1 wt % to about 40 wt % of the composition.
 46. Themethod of claim 34, wherein: the first compound is between about 0.1 wt% and about 30 wt % of the composition; and the second compound isbetween about 0.1 wt % to about 30 wt % of the composition.
 47. Themethod of claim 34, wherein: the first compound is between about 0.1 wt% and about 20 wt % of the composition; and the second compound isbetween about 0.1 wt % and about 20 wt % of the composition.
 48. Themethod of claim 34, wherein: the first compound is between about 0.1 wt% and about 10 wt % of the composition; and the second compound isbetween about 0.1 wt % and about 10 wt % of the composition.
 49. Themethod of claim 34, wherein: the first compound is between about 0.1mg/mL to about 70 mg/mL of the composition; and the second compound isbetween about 0.01% to about 50% the composition.
 50. The method ofclaim 34, wherein the first compound and the second compound are inlyophilized form.
 51. The method of claim 34, wherein the first compoundand the second compound are in hydrated form.
 52. The method of claim34, wherein the biocompatible matrix comprises one or more of hyaluronicacid, hyaluronates, lecithin gels, pluronics, poly(ethyleneglycol),poloxamers, chitosans, xyloglucans, collagens, fibrins, polyesters,poly(lactides), poly(glycolide), poly(lactic-co-glycolic acid (PLGA),sucrose acetate isobutyrate, glycerol monooleate, poly anhydrides, polycaprolactone sucrose, or glycerol monooleate or a combination thereof.53. The method of claim 34, wherein the biocompatible matrix comprises apoloxamer.
 54. The method of claim 34, wherein the poloxamer isPoloxamer
 407. 55. The method of claim 34, wherein the biocompatiblematrix comprises hyaluronic acid.
 56. The method of claim 34, whereinthe biocompatible matrix is a hydrogel.
 57. The method of claim 34,wherein the biocompatible matrix is a biocompatible gel or foam.
 58. Themethod of claim 34, wherein the composition is a controlled releaseformulation.
 59. The method of claim 58, wherein the controlled releaseformulation when administered to a subject intratympanically ortranstympanically imparts an immediate release, a delayed release, asustained release, an extended release, a variable release, a pulsatilerelease, or a bi-modal release of one or more of a) the GSK3β inhibitoror Wnt agonist, or a pharmaceutically acceptable salt thereof; and b)the Notch agonist or HDAC inhibitor, or a pharmaceutically acceptablesalt thereof.
 60. The method of claim 34, wherein the method produces apopulation of Lgr5⁺ cells that are in s-phase.
 61. The method of claim34, wherein administering intratympanically or transtympanically thecomposition to a subject results in improved auditory functioning of thesubject.
 62. The method of claim 34, wherein administering isintratympanically.
 63. The method of claim 34, wherein administering istranstympanically.